Hybrid immunoglobulin containing non-peptidyl linkage

ABSTRACT

The present invention provides compounds producing compounds having the structure (I). Wherein A is a first polypeptide component of the compound; wherein C is a second polypeptide component of the compound, which polypeptide component comprises consecutive amino acids which (i) are identical to a stretch of consecutive amino acids present in a chain of an Fc domain of an antibody; (ii) bind to an Fc receptor, and (iii) have at their N-terminus a sequence selected from the group consisting of a cysteine, selenocysteine, CP, CPXCP (where X=P, R, or S), CDKTHTCPPCP, CVECPPCP, CCVECPPCP and CDTPPPCPRCP, wherein B is a chemical structure linking A and C; wherein the dashed line between B and C represents a peptidyl linkage; wherein the solid line between A and B represents a nonpeptidyl linkage comprising the structure (II).

The present application is a § 371 national stage of PCT InternationalApplication No. PCT/US2014/029511, filed Mar. 14, 2014, claiming thebenefit of U.S. Provisional Patent Application No. 61/799,784, filedMar. 15, 2013, the contents of each of which are hereby incorporated byreference in their entirety.

REFERENCE TO A SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“181109_83134-PCT-US_SubstituteSequenceListing_DH.txt,” which is 272kilobytes in size, and which was created Nov. 9, 2018 in the IBM-PCmachine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Nov. 9, 2018 aspart of this application.

Throughout this application, various publications are referenced. Thedisclosures of all referenced publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Proteins prefer to form compact globular or fibrous structures,minimizing their exposure to solvent. This tendency is inherent both inthe polypeptide backbone with its propensity for hydrogen-bondedsecondary structure, and in side chain interactions that promotetertiary folding. Thus, previous efforts to introduce “flexibility” intoantibodies using peptides have been largely inadequate. For example, itis common to employ combinations of an amino acid that favors solventinteractions (e.g., serine) with one that breaks up helical structure(e.g., glycine). While this approach is useful in making fusion proteinssuch as single-chain antibody fragments (scFv), the resulting structuresare actually quite compact with no evidence of extendibility (forexample, see Robert et al, (2009) Engineered antibody interventionstrategies for Alzheimer's disease and related dementias by targetingamyloid and toxic oligomers. Protein Eng. Des. Sel. 22, 199-208).Furthermore, such sequences are likely to create additional problems dueto their intrinsic immunogenicity and proteolytic susceptibility.

There is a need for new protein compounds, incorporating nonproteinchains, that are both flexible and extendible, as well as processes forproducing such compounds.

SUMMARY OF THE INVENTION

The present invention provides a compound having the structure:

wherein A is a first polypeptide component of the compound;wherein C is a second polypeptide component of the compound, whichpolypeptide component comprises consecutive amino acids which (i) areidentical to a stretch of consecutive amino acids present in a chain ofan F_(c) domain of an antibody; (ii) bind to an F_(c) receptor; and(iii) have at their N-terminus a sequence selected from the groupconsisting of a cysteine, selenocysteine, CP, CPXCP (where X=P, R, or S)(SEQ ID NOs: 128-130), CDKTHTCPPCP (SEQ ID NO: 131), CVECPPCP (SEQ IDNO: 132), CCVECPPCP (SEQ ID NO: 133) and CDTPPPCPRCP (SEQ ID NO: 134),wherein B is a chemical structure linking A and C;wherein the dashed line between B and C represents a peptidyl linkage;wherein the solid line between A and B represents a nonpeptidyl linkagecomprising the structure:

wherein

is

in which R₅ is an alkyl or aryl groupwherein R₁ is H or is part of an additional structure that is a cyclicstructure, wherein the additional cyclic structure comprises R₁ or aportion of R₁, and may also comprise R₂ or a portion of R₂, and thecarbon between R₂ and the alkene double bond;with the proviso that if

is

R₃ is a H; if

is

is a triazole ring that comprises

and if

is

is a N-alkyl or aryl substituted isoxazoline ring that comprises

andwherein R₂ represents an organic structure which connects to one of A orB and R₄ represents an organic structure which connects to the other ofA or B.

The present invention provides a process for producing a compound havingthe structure:

wherein A is a first polypeptide component of the compound;wherein C is a second polypeptide component of the compound, whichpolypeptide component comprises consecutive amino acids which (i) areidentical to a stretch of consecutive amino acids present in a chain ofan F_(c) domain of an antibody; (ii) bind to an F_(c) receptor; and(iii) have at their N-terminus a sequence selected from the groupconsisting of a cysteine, selenocysteine, CP, CPXCP (where X=P, R, or S)(SEQ ID NOs: 128-130), CDKTHTCPPCP (SEQ ID NO: 131), CVECPPCP (SEQ IDNO: 132), CCVECPPCP (SEQ ID NO: 133) and CDTPPPCPRCP (SEQ ID NO: 134),wherein B is a chemical structure linking A and C;wherein the dashed line between B and C represents a peptidyl linkage;wherein the solid line between A and B represents a nonpeptidyl linkagecomprising the structure:

wherein

is

in which R₅ is an alkyl or aryl groupwherein R₁ is H or is part of an additional structure that is a cyclicstructure, wherein the additional cyclic structure comprises R₁ or aportion of R₁, and may also comprise R₂ or a portion of R₂, and thecarbon between R₂ and the alkene double bond;with the proviso that if

is

R₃ is a H; if

is

is a triazole ring that comprises

and if

is

is a N-alkyl or aryl substituted isoxazoline ring that comprises;

andwherein R₂ represents an organic structure which connects to one of A orB and R₄ represents an organic structure which connects to the other ofA or B;which comprises the following steps:a) obtaining an A′ which comprises A or a derivative of A, and a firstterminal reactive group;b) obtaining a B′ which comprises B or a derivative of B, a secondterminal reactive group and a third terminal reactive group, wherein thesecond terminal reactive group is capable of reacting with the firstterminal reactive group to form a non-peptidyl linkage;c) obtaining a C′ which comprises C or a derivative of C, and a fourthterminal reactive group, wherein the fourth terminal reactive group iscapable of reacting with the third terminal reactive group to form apeptidyl linkage; andd) reacting A′, B′ and C′ in any order to produce the compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the preparation of alkyne-modified TNR1B by cleavage of aTNR1B-intein fusion protein with cystyl-propargylamide. The inteinby-product is removed by chitin chromatography. Azide-modified TNR1B andcycloalkyne-modified TNR1B are similarly prepared usingcystyl-3-azidopropylamide, and various cyclooctyne (eg. DIBAC)derivatives of cysteine, respectively.

FIG. 2 shows the cleavage of TNR1B by (1) cysteine, (2)cysteine+mercaptoethane sulfonate (MESNA), (3) cystyl-propargylamide,(4) cystyl-propargylamide+MESNA, and (5) MESNA. All compounds were usedat 50 mM concentration.

FIG. 3 shows the preparation of azide-modified Fc6 by ligation(peptidyl) of the Fc6 dimer and azide-DKTHT-thioester (Table 1).

FIG. 4 shows the preparation of azide-modified Fc6 by ligation(peptidyl) of the Fc6 dimer and azide-PEG_(n)-DKTHT-thioester (Table 1).Cycloalkyne-modified Fc is similarly prepared by usingDIBAC-PEG₁₂-thioester.

FIG. 5 shows SDS-PAGE analysis (reducing conditions) of (1) unmodifiedFc6, (2) the Az-DKTHT-Fc6 reaction product of FIG. 3, and (3) theAz-PEG₄-DKTHT-Fc6 reaction product of FIG. 4.

FIG. 6 shows the synthesis of TNR1B-alkyne-azide-Fc6 by ligation(non-peptidyl) of alkyne-modified TNR1B and Az-DKTHT-Fc6.

FIG. 7 shows the synthesis of TNR1B-alkyne-azide-PEG_(n)-Fc6 by ligation(non-peptidyl) of alkyne-modified TNR1B and azide-PEG_(n)-DKTHT-Fc6. Inthis example, n=4.

FIG. 8 shows SDS-PAGE analysis (reducing conditions) of (1)alkyne-modified TNR1B alone, (2) alkyne-modified TNR1B+Az-DKTHT-Fc6 inthe absence of catalyst, (3) alkyne-modified TNR1B+Az-DKTHT-Fc6+catalystleading to the product of FIG. 6, and (4) dialyzed alkyne-modifiedTNR1B+Az-DKTHT-Fc6+catalyst leading to increased formation of theproduct of FIG. 6, (5) alkyne-modified TNR1B+Az-PEG₄-DKTHT-Fc6 in theabsence of catalyst, (6) alkyne-modifiedTNR1B+Az-PEG₄-DKTHT-Fc6+catalyst leading to the product of FIG. 7, and(7) dialyzed alkyne-modified TNR1B+Az-PEG₄-DKTHT-Fc6+catalyst leading toincreased formation of the product of FIG. 7. The arrows correspond to(a) Mr˜75,000, (b) Mr˜65,000, (c) Mr˜43,000, and (d) Mr˜28,000.

FIG. 9 shows SDS-PAGE analysis (reducing conditions) of (1) TNR1B-Fcfusion protein (etanercept) alone, (2) alkyne-modifiedTNR1B+Az-DKTHT-Fc6+catalyst leading to the product of FIG. 6, (3)TNR1B-Fc fusion protein (etanercept), and (4) alkyne-modifiedTNR1B+Az-PEG₄-DKTHT-Fc6 leading to the product of FIG. 7. The arrowscorrespond to (a) Mr˜75,000, (b) Mr˜65,000, (c) Mr˜43,000, and (d)Mr˜28,000.

FIG. 10 shows SDS-PAGE analysis (reducing conditions) of (1) unmodifiedFc6+catalyst, (2) alkyne-modified TNR1B+unmodified Fc6+catalyst (3)Az-DKTHT-Fc6+catalyst, (4) alkyne-modified TNR1B+Az-DKTHT-Fc6+catalystleading to the product of FIG. 6, and (5) alkyne-modified TNR1B alone.The arrows correspond to (a) Mr˜75,000, (b) Mr˜65,000, (c) Mr˜43,000,(d) Mr˜28,000, and (e) Mr˜27,000.

FIG. 11 shows tryptic peptided identified by LC/MS in theTNR1B-alkyne-azide-DKTHT-Fc6 product (Mr˜75,000) of FIG. 10. Theunderlined peptide sequences are those identified by LC/MS that arederived from the parent TNR1B (upper) and Fc6 (lower) sequences.

FIG. 12 shows SPR analysis of TNF-α binding by theTNR1B-alkyne-azide-DKTHT-Fc6 (left panel) andTNR1B-alkyne-azide-PEG₄-DKTHT-Fc6 (right panel) reaction products ofFIG. 9. The kinetic binding data are summarized in Table 2.

FIG. 13 shows the preparation of adalimumab Fab′ in a three-stepprocess: 1) IdeS cleavage to the Fab′2+Fc′ fragments, 2) Protein Achromatography to remove the Fc′ fragment, and 3) mild reduction of theFab′2 fragment to the Fab′ fragment with 2-mercaptoethylamine (MEA).

FIG. 14 shows SDS-PAGE analysis of (1) adalimumab, (2) adalimumab afterIdeS cleavage, (3) adalimumab Fab′2 after Protein A purification, (4)adalimumab Fab′ after MEA treatment of the Protein A purified Fab′2, (5)adalimumab Fab′2 after Protein A purification, and (6) adalimumab Fab′after MEA treatment of the Protein A purified Fab′2. The samples inlanes 1, 2, 5 and 6 were analysis under reducing conditions; while thesamples in lanes 3 and 4 were analyzed under non-reducing conditions.The arrows correspond to the (a) heavy chain, (b) heavy chain Fc′fragment, (c) heavy chain Fd′ (variable region-containing) fragment, and(d) light chain.

FIG. 15 shows the preparation of cycloalkyne-modified Fab′ by thereaction of adalimumab Fab′ with DIBAC-PEG_(y)-Lys (Mal). In thisexample, PEGy=PEG₁₂.

FIG. 16 shows SDS-PAGE analysis (non-reducing conditions) of thesynthesis and purification of cycloalkyne-modified adalimumab Fab′.Upper panel shows the reaction at (1-6) pH 7.4 and (7-12) pH 7.0. TheDIBAC-PEG_(y)-Lys(Mal) to Fab′ ration was (1) 0, (2) 10:1, (3) 5:1, (4)2.5:1, (5) 1.2:1, (6) 0.6:1, (7) 0, (8) 10, (9) 5, (10) 2.5, (11) 1.2,and (12) 0.6:1. The lower panel shows (1) unreacted Fab′, (2) through(12) Protein L flow-through fractions containing only thecycloalkyne-modified Fab′.

FIG. 17 shows SDS-PAGE analysis (reducing conditions) of (1) Fc6, (2)Az-DKTHT-Fc6, (3) Az-PEG₁₂-DKTHT-Fc6, (4) Az-PEG₂₄-DKTHT-Fc6, and (5)Az-PEG₃₆-DKTHT-Fc6.

FIG. 18 shows size-exclusion chromatography of (a) Az-PEG₃₆-DKTHT-Fc6,(b) Az-PEG₂₄-DKTHT-Fc6, (c) Az-PEG₁₂-DKTHT-Fc6, (d) Az-DKTHT-Fc6, and(e) Fc6.

FIG. 19 shows the synthesis of Fab′-PEGy-alkyne-azide-PEGx-Fc6 byligation (non-peptidyl) of cycloalkyne-modified adalimumab Fab′ andazide-modified Fc6.

FIG. 20 shows the Fab′-PEGy-alkyne-azide-PEGx-Fc6 product series.

FIG. 21 shows SDS-PAGE analysis of (1) adalimumab whole antibody, (2)adalimumab Fab′, (3) Fab′-PEG₁₂-alkyne, (4)Fab′-PEG₁₂-alkyne+Az-DKTHT-Fc6, (5) Az-DKTHT-Fc6, (6)Fab′-PEG₁₂-alkyne+Az-PEG₁₂-DKTHT-Fc6, (7) Az-PEG₁₂-DKTHT-Fc6, (8)Fab′-PEG₁₂-alkyne+Az-PEG₂₄-DKTHT-Fc6, (9) Az-PEG₂₄-DKTHT-Fc6 alone, (10)Fab′-PEG₁₂-alkyne+Az-PEG₃₆-DKTHT-Fc6, (11) Az-PEG₃₆-DKTHT-Fc6, and (12)Fc6. Samples were run under reducing conditions (upper panel) andnon-reducing conditions (lower panel). In the upper panel the arrowshows (a) Fab′-PEGy-alkyne-azide-PEGx-Fc6 heavy chain. In the lowerpanels the arrows show (a) two-handed Fab′-PEGy-alkyne-azide-PEGx-Fc6molecules, and (b) one-handed Fab′-PEGy-alkyne-azide-PEGx-Fc6 molecules.

FIG. 22 shows size-exclusion chromatography (SEC) of two-handed reactionproducts: (a) Fab′-PEG₁₂-alkyne-azide-PEG₃₆-DKTHT-Fc6, (b)Fab′-PEG₁₂-alkyne-azide-PEG₂₄-DKTHT-Fc6, (c)Fab′-PEG₁₂-alkyne-azide-PEG₁₂-DKTHT-Fc6, (d)Fab′-PEG₁₂-alkyne-azide-DKTHT-Fc6, and (e) whole adalimumab.

FIG. 23 shows the inhibition of TNF-α cytotoxity on WEHI cells byreaction products. The upper panel shows the (a) Fc6 control, (b)cycloalkyne-modified Fab′, (c) Fab′-PEG₁₂-alkyne-azide-DKTHT-Fc6, and(d) Fab′-PEG₁₂-alkyne-azide-PEG₁₂-DKTHT-Fc6. The lower panel shows (a)Fc6 control, (b) cycloalkyne-modified Fab′, (c)Fab′-PEG₁₂-alkyne-azide-PEG₂₄-DKTHT-Fc6, and (d)Fab′-PEG₁₂-alkyne-azide-PEG₃₆-DKTHT-Fc6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound having the structure:

wherein A is a first polypeptide component of the compound;wherein C is a second polypeptide component of the compound, whichpolypeptide component comprises consecutive amino acids which (i) areidentical to a stretch of consecutive amino acids present in a chain ofan F_(c) domain of an antibody; (ii) bind to an F_(c) receptor; and(iii) have at their N-terminus a sequence selected from the groupconsisting of a cysteine, selenocysteine, CP, CPXCP (where X=P, R, or S)(SEQ ID NOs: 128-130), CDKTHTCPPCP (SEQ ID NO: 131), CVECPPCP (SEQ IDNO: 132), CCVECPPCP (SEQ ID NO: 133) and CDTPPPCPRCP (SEQ ID NO: 134),wherein B is a chemical structure linking A and C;wherein the dashed line between B and C represents a peptidyl linkage;wherein the solid line between A and B represents a nonpeptidyl linkagecomprising the structure:

wherein

is

in which R₅ is an alkyl or aryl group

-   -   wherein R₁ is H or is part of an additional structure that is a        cyclic structure, wherein the additional cyclic structure        comprises R₁ or a portion of R₁, and may also comprise R₂ or a        portion of R₂, and the carbon between R₂ and the alkene double        bond;    -   with the proviso that if

-   -    is

-   -    R₃ is a H; if

-   -    is

-   -    is a triazole ring that comprises

-   -    and if

-   -    is

-   -    is a N-alkyl or aryl substituted isoxazoline ring that        comprises

-   -    and        wherein R₂ represents an organic structure which connects to one        of A or B and R₄ represents an organic structure which connects        to the other of A or B.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

wherein R₁ is H or is part of an additional structure that is a cyclicstructure, wherein the additional cyclic structure comprises R₁ or aportion of R₁, and may also comprise R₂ or a portion of R₂, and thecarbon between R₂ and the alkene double bond.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

wherein R₁ is H or is part of an additional structure that is a cyclicstructure, wherein the additional cyclic structure comprises R₁ or aportion of R₁, and may also comprise R₂ or a portion of R₂, and thecarbon between R₂ and the alkene double bond.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

wherein R₁ is part of an additional structure that is a cyclicstructure, wherein the additional cyclic structure comprises R₁ or aportion of R₁, and may also comprise R₂ or a portion of R₂, and thecarbon between R₂ and the alkene double bond.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

wherein R₁ is part of an additional structure that is a cyclicstructure, wherein the additional cyclic structure comprises R₁ or aportion of R₁, and may also comprise R₂ or a portion of R₂, and thecarbon between R₂ and the alkene double bond.

In some embodiments, R₁ and R₂ are linked via at least one direct bondso as to form a cyclic structure comprising

i) a portion of R₁,ii) a portion of R₂,iii) the carbon between R₂ and the alkene double bond, andiv) the alkene double bond.

In some embodiments, R₁ is selected from the group consisting of:

which is optionally substituted at any position.

In some embodiments, R₁ is

which is optionally substituted at any position.

In some embodiments, R₁ is

which is optionally substituted at any position.

In some embodiments, R₁ is

which is optionally substituted at any position.

In some embodiments, the carbon between R₂ and the alkene double bondis:

(i) directly bonded to R₂ with a single bond and substituted with twosubstituents independently selected from the group consisting ofhydrogen, halogen, optionally substituted benzyl, optionally substitutedalkyl or optionally substituted alkoxy; or(ii) directly bonded to R₂ via a double bond and a single bond.

In some embodiments, the carbon between R₂ and the alkene double bond issubstituted with two hydrogens and directly bonded to R₂ with a singlebond.

In some embodiments, the carbon between R₂ and the alkene double bond isdirectly bonded to R₂ via a double bond and a single bond.

In some embodiments, the carbon between R₂ and the alkene double bond isdirectly bonded to R₂ via a double bond and a single bond so as to forma phenyl ring which is optionally substituted at any position.

In some embodiments, R₂ is

wherein R₂ is attached to A via J, andwherein J is a bond or an organic structure comprising or consisting ofa chain of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties selected from thegroup consisting of a [PEG(y)]z, polyalkylene glycol, polyoxyalkylatedpolyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid),poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₄alkyl, amine, sulfur, oxygen, succinimide, maleimide, glycerol,triazole, isoxazolidine, C₁-C₄ acyl, succinyl, malonyl, glutaryl,phthalyl, adipoyl and an amino acid,wherein [PEG(y)]z is:

wherein y=1-100 and z=1-10.

In some embodiments, R₂ is

wherein R₂ is attached to A via J, andwherein R₂ is attached to R₁ via the nitrogen atom of R₂, andwherein J is a bond or an organic structure comprising or consisting ofa chain of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties selected from thegroup consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylatedpolyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid),poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₄alkyl, amine, sulfur, oxygen, succinimide, maleimide, glycerol,triazole, isoxazolidine, C₁-C₄ acyl, succinyl, malonyl, glutaryl,phthalyl, adipoyl and an amino acid,wherein [PEG(y)]z is:

wherein y=1-100 and z=1-10.

In some embodiments, R₂ is

-   -   which is optionally substituted at any position,        wherein R₂ is attached to R₁ via the nitrogen atom of R₂, and        wherein J is a bond or an organic structure comprising or        consisting of a chain of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more        moieties selected from the group consisting of [PEG(y)]z,        polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol,        polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic        acid), polysaccharide, a branched residue, C₁-C₄ alkyl, amine,        sulfur, oxygen, succinimide, maleimide, glycerol, triazole,        isoxazolidine, C₁-C₄ acyl, succinyl, malonyl, glutaryl,        phthalyl, adipoyl and an amino acid,        wherein [PEG(y)]z is:

wherein y=1-100 and z=1-10.

In some embodiments, R₂ is

which is optionally substituted at any position.

In some embodiments, R₂ is

which is optionally substituted at any position.

In some embodiments, R₂ is

which is optionally substituted at any position.

In some embodiments, R₂ is

which is optionally substituted at any position.

In some embodiments, R₂ is

which is optionally substituted at any position.

In some embodiments, R₂ is

which is optionally substituted at any position.

In some embodiments, R₁ and R₂ taken together are:

which is optionally substituted at any position,wherein J is a bond or an organic structure comprising or consisting ofa chain of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties selected from thegroup consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylatedpolyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid),polylactic-glycolic acid), polysaccharide, a branched residue, C₁-C₄alkyl, amine, sulfur, oxygen, succinimide, maleimide, glycerol,triazole, isoxazolidine, C₁-C₄ acyl, succinyl, malonyl, glutaryl,phthalyl, adipoyl and an amino acid,wherein [PEG(y)]z is:

wherein y=1-100 and z=1-10.

In some embodiments, R₁ and R₂ taken together are

which is optionally substituted at any position.

In some embodiments, R₁ and R₂ taken together are

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

which is optionally substituted at any position,wherein J is a bond or an organic structure comprising or consisting ofa chain of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more moieties selected from thegroup consisting of [PEG(y)]z, polyalkylene glycol, polyoxyalkylatedpolyol, polyvinyl alcohol, polyvinyl alkyl ether, poly(lactic acid),poly(lactic-glycolic acid), polysaccharide, a branched residue, C₁-C₄alkyl, amine, sulfur, oxygen, succinimide, maleimide, glycerol,triazole, isoxazolidine, C₁-C₄ acyl, succinyl, malonyl, glutaryl,phthalyl, adipoyl and an amino acid,wherein [PEG(y)]z is:

wherein y=1-100 and z=1-10.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   which is optionally substituted at any position,        wherein J is a bond or an organic structure comprising or        consisting of a chain of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more        moieties selected from the group consisting of [PEG(y)]z,        polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol,        polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic        acid), polysaccharide, a branched residue, C₁-C₄ alkyl, amine,        sulfur, oxygen, succinimide, maleimide, glycerol, triazole,        isoxazolidine, C₁-C₄ acyl, succinyl, malonyl, glutaryl,        phthalyl, adipoyl and an amino acid,    -   wherein [PEG(y)]z is:

-   -   wherein y=1-100 and z=1-10.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   which is optionally substituted at any position,        wherein J is a bond or an organic structure comprising or        consisting of a chain of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more        moieties selected from the group consisting of [PEG(y)]z,        polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol,        polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic        acid), polysaccharide, a branched residue, C₁-C₄ alkyl, amine,        sulfur, oxygen, succinimide, maleimide, glycerol, triazole,        isoxazolidine, C₁-C₄ acyl, succinyl, malonyl, glutaryl,        phthalyl, adipoyl and an amino acid,    -   wherein [PEG(y)]z is:

-   -   wherein y=1-100 and z=1-10.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   which is optionally substituted at any position,        wherein J is a bond or an organic structure comprising or        consisting of a chain of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more        moieties selected from the group consisting of [PEG(y)]z,        polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol,        polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic        acid), polysaccharide, a branched residue, C₁-C₄ alkyl, amine,        sulfur, oxygen, succinimide, maleimide, glycerol, triazole,        isoxazolidine, C₁-C₄ acyl, succinyl, malonyl, glutaryl,        phthalyl, adipoyl and an amino acid,    -   wherein [PEG(y)]z is:

-   -   wherein y=1-100 and z=1-10.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   which is optionally substituted at any position.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

In some embodiments, R₁ is H.

In some embodiments, J is an organic structure comprising a [PEG(y)]zgroup.

In some embodiments, J is an organic structure comprising a polyalkyleneglycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkylether, poly(lactic acid), poly(lactic-glycolic acid), or polysaccharidegroup.

In some embodiments, J is an organic structure comprising a C₁-C₄ alkylgroup.

In some embodiments, J is an organic structure comprising a succinimide.

In some embodiments, J is an organic structure comprising an amine.

In some embodiments, J is an organic structure comprising a succinyl,malonyl, glutaryl, phthalyl or adipoyl.

In some embodiments, J is an organic structure comprising a malonyl.

In some embodiments, J is an organic structure comprising an amino acid.

In some embodiments, J is an organic structure comprising a cysteine.

In some embodiments, J is an organic structure comprising a lysine.

In some embodiments, J is an organic structure consisting of a chain of3 moieties selected from the group consisting of [PEG(y)]z, polyalkyleneglycol, polyoxyalkylated polyol, polyvinyl alcohol, polyvinyl alkylether, poly(lactic acid), poly(lactic-glycolic acid), polysaccharide,C₁-C₄ alkyl, amine, sulfur, oxygen, succinimide, maleimide, glycerol,triazole, isoxazolidine, C1-C4 acyl, succinyl, malonyl, glutaryl,phthalyl, adipoyl or an amino acid.

In some embodiments, J is an organic structure consisting of a chain offour moieties selected from the group consisting of [PEG(y)]z,polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol,polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid),polysaccharide, C₁-C₄ alkyl, amine, sulfur, oxygen, succinimide,maleimide, glycerol, triazole, isoxazolidine, C₁-C₄ acyl, succinyl,malonyl, glutaryl, phthalyl, adipoyl or an amino acid.

In some embodiments, J is an organic structure consisting of a chain offive moieties selected from the group consisting of [PEG(y)]z,polyalkylene glycol, polyoxyalkylated polyol, polyvinyl alcohol,polyvinyl alkyl ether, poly(lactic acid), poly(lactic-glycolic acid),polysaccharide, C₁-C₄ alkyl, amine, sulfur, oxygen, succinimide,maleimide, glycerol, triazole, isoxazolidine, C₁-C₄ acyl, succinyl,malonyl, glutaryl, phthalyl, adipoyl or an amino acid.

In some embodiments, J comprises a [PEG(y)]z group bonded to a lysine.

In some embodiments, J comprises a C₁-C₄ acyl group bonded to asuccinimide group.

In some embodiments, J comprises a lysine bonded to a C₁-C₄ acyl.

In some embodiments, J comprises a [PEG(y)]z group, which is bonded to aglutaryl.

In some embodiments, J is an organic structure consisting of a chain offive moieties selected from the group consisting of [PEG(y)]z,succinimide, C₁-C₄ acyl, glutaryl or lysine.

In some embodiments, J is a bond.

In some embodiments, J is a cysteine.

In some embodiments, J has the structure:

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, J has a linear structure.

In some embodiments, J has a branched structure.

In some embodiments, R₂ is

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, R₂ is

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, R₂ is

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, R₁ and R₂ taken together are:

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, R₁ and R₂ taken together are:

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, R₁ and R₂ taken together are:

wherein n 1-3, m is 1-4, y is 1-100 and z is 1-10.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   wherein [PEG(y)]z is:

-   -   wherein y=1-100 and z=1-10.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

-   -   wherein [PEG(y)]z is

-   -   wherein y=1-100 and z=1-10;    -   wherein [PEG(x)]w is:

-   -   wherein x=1-100 and w=1-10.

In some embodiments, y is 1-20.

In some embodiments, y is 21-40.

In some embodiments, y is 41-60.

In some embodiments, y is 61-80.

In some embodiments, y is 30-50

In some embodiments, y is 12, 24, 36 or 48.

In some embodiments, z is 1.

In some embodiments, z is 0.

In some embodiments, the terminal carbonyl is of the [PEG(y)]z group ispart of an amide bond.

In some embodiments, the terminal amine of the [PEG(y)]z group is partof an amide bond.

In some embodiments, R₄ is

wherein x is 1-100, and w is 0-5.

In some embodiments, x is 1-20.

In some embodiments, x is 21-40.

In some embodiments, x is 41-60.

In some embodiments, x is 61-80.

In some embodiments, x is 30-50

In some embodiments, x is 12, 24, 36 or 48.

In some embodiments, w is 1.

In some embodiments, w is 0.

In some embodiments, R₄ has the structure:

In some embodiments, R₄ is attached to B via the terminal carbonylcarbon.

In some embodiments, the solid line between A and B represents anonpeptidyl linkage comprising the structure:

wherein p=0-5, 0-10, 0-50, or 0-100.

In some embodiments, R₂ is attached to A via a carbon-nitrogen bond or acarbon-sulfur bond.

In some embodiments, R₂ is attached to A via a carbon-nitrogen bond.

In some embodiments, the carbon-nitrogen bond is an amide bond.

In some embodiments, R₂ is attached to A via an amide bond between theC-terminal amino acid of A and an amino group in B.

In some embodiments, the terminal amino acid is cysteine.

In some embodiments, R₂ is attached to A via a carbon-sulfur bond.

In some embodiments, R₂ is attached to A via a carbon-sulfur bond formedbetween R₂ and a free thiol.

In some embodiments, R₂ is attached to A via a succinimide-sulfur bond.

In some embodiments, J comprises a branched residue.

In some embodiments, J is attached to more than one A via the branchedresidue.

In some embodiments, B comprises a branched residue.

In some embodiments, B is linked to more than one A, each via anonpeptidyl linkage with the branched residue.

In some embodiments, B is an organic acid residue.

In some embodiments, B is a stretch of 1-50 amino acid residues, andoptionally, an organic acid residue.

In some embodiments, B is a stretch of 1-10 consecutive amino acids.

In some embodiments, B comprises a stretch of consecutive amino acids inthe sequence, or a portion thereof, EPKSCDKTHTCPPCP (SEQ ID NO: 135),ERKCCVECPPCP (SEQ ID NO: 136), ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQID NO: 137), ESKYGPPCPSCP (SEQ ID NO: 138).

In some embodiments, B has a threonine at its C-terminus.

In some embodiments, B is linked to C via a peptidyl linkage between theN-terminal cysteine or selenocysteine of C and an amino acid residue oran organic acid residue of B.

In some embodiments, C is a second polypeptide component of thecompound, which polypeptide component comprises consecutive amino acidswhich (i) are identical to a stretch of consecutive amino acids presentin a chain of an F_(c) domain of an antibody; (ii) bind to an F_(c)receptor; and (iii) have at their N-terminus a sequence comprising anaturally occurring cysteine selected from the group consisting of CP,CPXCP (where X=P, R, or S) (SEQ ID NOs: 128-130), CDKTHTCPPCP (SEQ IDNO: 131), CVECPPCP (SEQ ID NO: 132), CCVECPPCP (SEQ ID NO: 133) andCDTPPPCPRCP (SEQ ID NO: 134).

In some embodiments, C is a second polypeptide component of thecompound, which polypeptide component comprises consecutive amino acidswhich (i) are identical to a stretch of consecutive amino acids presentin a chain of an F_(c) domain of an antibody; (ii) bind to an F_(c)receptor; and (iii) have at their N-terminus a sequence comprising anon-naturally occurring cysteine or selenocysteine.

In some embodiments, C comprises consecutive amino acids which areidentical to a stretch of consecutive amino acids present in the chainof an Fc domain of an antibody selected from the group consisting ofIgG, IgM, IgA, IgD, and IgE.

In some embodiments, C comprises consecutive amino acids which areidentical to a stretch of consecutive amino acids present in the chainof an Fc6 domain of an antibody.

In some embodiments, A comprises a secreted protein.

In some embodiments, A comprises an extracellular domain of a protein.

In some embodiments, A has biological activity.

In some embodiments, the biological activity is target-binding activity.

In some embodiments, the A is an independently-folding protein or aportion thereof.

In some embodiments, A is a glycosylated protein.

In some embodiments, A comprises intra-chain disulfide bonds.

In some embodiments, A binds a cytokine.

In some embodiments, the cytokine is TNFα.

In some embodiments, A comprises at least one stretch of consecutiveamino acids which are identical to a stretch of consecutive amino acidspresent in the heavy chain of a Fab or a Fab′ of an antibody.

In some embodiments, A comprises at least one at least one stretch ofconsecutive amino acids which are identical to a stretch of consecutiveamino acids present in the light chain of a Fab or a Fab′ of anantibody.

In some embodiments, A comprises at least one Fab or Fab′ of anantibody, or a portion of the at least one Fab or Fab′.

In some embodiments, A comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10copies of the Fab or Fab′ or portion thereof.

In some embodiments, A comprises Fab-1 or Fab′1, or a portion thereof ofthe antibody.

In some embodiments, A comprises Fab-2 or Fab′2, or a portion thereof ofthe antibody.

In some embodiments, A comprises two Fab or Fab′ hands of the antibody.

In some embodiments, the Fab or Fab′ is present in adalimumab. In someembodiments, A comprises at least one stretch of consecutive amino acidswhich are identical to a stretch of consecutive amino acids present in asingle chain antibody.

In some embodiments, A comprises at least one stretch of consecutiveamino acids which are identical to a stretch of consecutive amino acidspresent in a TNFα receptor.

In some embodiments, the TNFα receptor is TNR1B.

In some embodiments, the compound forms part of a homodimer.

In some embodiments, the compound forms part of a heterodimer.

The present invention provides a homodimer comprising a compound of theinvention.

The present invention provides a heterodimer comprising a compound ofthe invention.

In some embodiments, each compound of the dimer is capable of binding tothe other by at least one disulfide bond.

In some embodiments, each compound of the dimer is capable of binding tothe other by at least one disulfide bond between the C of each compound.

In some embodiments, each compound of the dimer is bound to the other byat least one disulfide bond.

In some embodiments, each compound of the dimer is bound to the other byat least one disulfide bond between the C of each compound.

In some embodiments, each compound of the dimer is non-covalently boundto the other.

The present invention provides a process for producing a compound havingthe structure:

wherein A is a first polypeptide component of the compound;wherein C is a second polypeptide component of the compound, whichpolypeptide component comprises consecutive amino acids which (i) areidentical to a stretch of consecutive amino acids present in a chain ofan F_(c) domain of an antibody; (ii) bind to an F_(c) receptor; and(iii) have at their N-terminus a sequence selected from the groupconsisting of a cysteine, selenocysteine, CP, CPXCP (where X=P, R, or S)(SEQ ID NOs: 128-130), CDKTHTCPPCP (SEQ ID NO: 131), CVECPPCP (SEQ IDNO: 132), CCVECPPCP (SEQ ID NO: 133) and CDTPPPCPRCP (SEQ ID NO: 134),wherein B is a chemical structure linking A and C;wherein the dashed line between B and C represents a peptidyl linkage;wherein the solid line between A and B represents a nonpeptidyl linkagecomprising the structure:

wherein

is

in which R₅ is an alkyl or aryl groupwherein R₁ is H or is part of an additional structure that is a cyclicstructure, wherein the additional cyclic structure comprises R₁ or aportion of R₁, and may also comprise R₂ or a portion of R₂, and thecarbon between R₂ and the alkene double bond;with the proviso that if

is

R₃ is a H;

if

is

is a triazole ring that comprises

and if

is

is a N-alkyl or aryl substituted isoxazoline ring that comprises

andwherein R₂ represents an organic structure which connects to one of A orB and R₄ represents an organic structure which connects to the other ofA or B;which comprises the following steps:a) obtaining an A′ which comprises A or a derivative of A, and a firstterminal reactive group;b) obtaining a B′ which comprises B or a derivative of B, a secondterminal reactive group and a third terminal reactive group, wherein thesecond terminal reactive group is capable of reacting with the firstterminal reactive group to form a non-peptidyl linkage;c) obtaining a C′ which comprises C or a derivative of C, and a fourthterminal reactive group, wherein the fourth terminal reactive group iscapable of reacting with the third terminal reactive group to form apeptidyl linkage; andd) reacting A′, B′ and C′ in any order to produce the compound.

In some embodiments, step d) is performed by first reacting A′ and B′ toproduce

wherein B″ comprises B and the third terminal reactive group, and thesolid line between B″ and A represents a non-peptidyl linkage; and thenreacting

with C′ to produce the compound.

In some embodiments, step d) is performed by first reacting C′ and B′ toproduce

wherein B″ comprises B and the second terminal reactive group, and thedashed line between B″ and C represents a peptidyl linkage; and thenreacting

with A′ to produce the compound.

In some embodiments, the first terminal reactive group is an azide, athiol, a nitrone or an alkyne.

In some embodiments, the first terminal reactive group is an alkyne.

In some embodiments, the alkyne is a cycloalkyne. In some embodiments,the alkyne is an eight-membered ring.

In some embodiments, the alkyne is an azacyclooctyne.

In some embodiments, the cycloalkyne is a biarylazacyclooctyne.

In some embodiments, the cycloalkyne is a cyclooctyne.

In some embodiments, the alkyne is a terminal alkyne.

In some embodiments, the first terminal reactive group is an azide,thiol or nitrone.

In some embodiments, the first terminal reactive group is an azide.

In some embodiments, the first terminal reactive group is a thiol.

In some embodiments, the first terminal reactive group is a nitrone.

In some embodiments, the first terminal reactive group is an N-alkylnitrone.

In some embodiments, the first terminal reactive group is an N-arylnitrone.

In some embodiments, the second terminal reactive group is an azide, athiol, a nitrone or an alkyne.

In some embodiments, the second terminal reactive group is an alkyne.

In some embodiments, the alkyne is a cycloalkyne. In some embodiments,the alkyne is an eight-membered ring.

In some embodiments, the alkyne is an azacyclooctyne.

In some embodiments, the cycloalkyne is a biarylazacyclooctyne.

In some embodiments, the cycloalkyne is a cyclooctyne.

In some embodiments, the alkyne is a terminal alkyne.

In some embodiments, the second terminal reactive group is an azide,thiol or nitrone.

In some embodiments, the second terminal reactive group is an azide.

In some embodiments, the second terminal reactive group is a thiol.

In some embodiments, the second terminal reactive group is a nitrone.

In some embodiments, the second terminal reactive group is an N-alkylnitrone.

In some embodiments, the second terminal reactive group is an N-arylnitrone.

In some embodiments, the first terminal reactive group is a terminalalkyne and the second terminal reactive group is an azide, thiol ornitrone.

In some embodiments, the second terminal reactive group is an azide.

In some embodiments, the second terminal reactive group is a thiol.

In some embodiments, the second terminal reactive group is a nitrone.

In some embodiments, the nitrone is an N-alkyl or N-aryl nitrone.

In some embodiments, the first terminal reactive group is an azide,thiol or nitrone, and the second terminal reactive group is a terminalalkyne.

In some embodiments, the first terminal reactive group is an azide.

In some embodiments, the first terminal reactive group is a thiol.

In some embodiments, the first terminal reactive group is a nitrone.

In some embodiments, the nitrone is an N-alkyl or N-aryl nitrone.

In some embodiments, the first terminal reactive group is a cycloalkyneand the second terminal reactive group is an azide, thiol or nitrone.

In some embodiments, the first terminal reactive group is an azide.

In some embodiments, the first terminal reactive group is a thiol.

In some embodiments, the first terminal reactive group is a nitrone.

In some embodiments, the nitrone is an N-alkyl or N-aryl nitrone.

In some embodiments, the first terminal reactive group is an azide,thiol or nitrone, and the second terminal reactive group is acycloalkyne.

In some embodiments, the first terminal reactive group is an azide.

In some embodiments, the first terminal reactive group is a thiol.

In some embodiments, the first terminal reactive group is a nitrone.

In some embodiments, the nitrone is an N-alkyl or N-aryl nitrone.

In some embodiments, the cycloalkyne is an eight-membered ring.

In some embodiments, the alkyne is an azacyclooctyne.

In some embodiments, the cycloalkyne is a biarylazacyclooctyne.

In some embodiments, the cycloalkyne is a cyclooctyne.

In some embodiments, the first terminal reactive group is an azide andthe second terminal reactive group is a terminal alkyne; or the firstterminal reactive group is an azide and the second terminal reactivegroup is a cycloalkyne; or the first terminal reactive group is a thioland the second terminal reactive group is a cycloalkyne; or the firstterminal reactive group is a N-alkyl nitrone or N-aryl nitrone and thesecond terminal reactive group is a cyclooctyne.

In some embodiments, the second terminal reactive group is an azide andthe first terminal reactive group is a terminal alkyne; or the secondterminal reactive group is an azide and the first terminal reactivegroup is a cycloalkyne; or the second terminal reactive group is a thioland the first terminal reactive group is a cycloalkyne; or the secondterminal reactive group is a N-alkyl nitrone or N-aryl nitrone and thefirst terminal reactive group is a cyclooctyne.

In some embodiments, the first terminal reactive group and the secondterminal reactive group react to produce a triazole, thiolene, N-alkylisoxazoline or N-aryl isoxazoline.

In some embodiments, the first terminal reactive group and the secondterminal reactive group react to produce a triazole.

In some embodiments, the first terminal reactive group and the secondterminal reactive group react to produce a thiolene.

In some embodiments, the first terminal reactive group and the secondterminal reactive group react to produce a N-alkyl isoxazoline or N-arylisoxazoline.

In some embodiments, the the third reactive group and the fourthterminal reactive group are each independently an amino acid or aminoacid derivative.

In some embodiments, the third reactive group is a threonine orthreonine derivative.

In some embodiments, the third reactive group is a thioester derivativeof an amino acid.

In some embodiments, the fourth reactive group is cysteine,selenocysteine, homocysteine, or homoselenosysteine, or a derivative ofcysteine, selenocysteine, homocysteine, or homoselenosysteine.

In some embodiments, the fourth reactive group is cysteine or aderivative of cysteine.

In some embodiments, the fourth reactive group is cysteine.

In some embodiments, A′ is prepared by the following steps:

-   -   i) obtaining an A″ which comprises A or a derivative of A, and a        stretch of consecutive amino acids comprising an intein;    -   ii) obtaining a substituted cysteine, selenocysteine,        homocysteine, or homoselenosysteine residue, or a substituted        derivative of a cysteine, selenocysteine, homocysteine, or        homoselenosysteine residue, wherein the cysteine residue is        substituted at the C-terminus with an organic structure        containing an alkyne, an azide, a thiol, or a nitrone; and    -   iii) reacting A″ with the substituted cysteine residue to        produce A′.

In some embodiments, the organic structure containing an alkyne isN-propargyl amine.

In some embodiments, A′ is prepared by the following steps:

-   -   i) obtaining an A″ which comprises A or a derivative of A, and        which comprises at least one free thiol group;    -   ii) obtaining a compound which comprises a first terminal        reactive group and a terminal maleimide; and    -   iii) reacting A″ with the compound of step ii) to produce A′.

In some embodiments, A″ is prepared by the following steps:

-   -   a) obtaining an A′″, wherein A′″ is a polypeptide which        comprises A or a derivative of A, and which comprises at least        one disulfide bond; and    -   b) treating A′″ with mercaptoethylamine (MEA) to produce A″.

In some embodiments, the A′″ is prepared by the following steps:

-   -   a) obtaining a monoclonal antibody which comprises A or        derivative of A, and which comprises at least one disulfide        bond; and    -   b) treating the polypeptide of step a) with IdeS to produce A′″.

In some embodiments, the monoclonal antibody binds TNFα.

In some embodiments, the monoclonal antibody is adalimumab.

In some embodiments, the compound according to any one of claims 1-146is produced.

In some embodiments, if R₁ is hydrogen and the first terminal reactivegroup is alkyne, then in step d) B′ is reacted in the presence of ametal catalyst.

In some embodiments, if R₁ is hydrogen and the second terminal reactivegroup is alkyne, then in step d) B′ is reacted in the presence of ametal catalyst.

In some embodiments, the metal catalyst is Ag(I) or Cu(I).

In some embodiments, A′ comprises one or more branched residue, whereineach branched residue comprises an additional first terminal reactivegroup.

In some embodiments, B′ comprises one or more branched residue, whereineach branched residue comprises an additional second terminal reactivegroup.

In some embodiments, B′ comprises one or more branched residue, whereineach branched residue comprises an additional third terminal reactivegroup.

In some embodiments, the branched residue is an amino acid residue.

In some embodiments, the amino acid residue is a lysine or a lysinederivative, arginine or an arginine derivative, aspartic acid or anaspartic acid derivative, glutamic acid or a glutamic acid derivative,asparagines or a asparagines derivative, glutamine or glutaminederivative, tyrosine or tyrosine derivative, cysteine or cysteinederivative or ornithine or ornithine derivative.

In some embodiments, the amino acid residue is substituted at theN-position with a residue containing a terminal amino or carbonyreactive group.

In some embodiments, the branched residue is an organic residuecontaining two or more terminal amino groups or two or more terminalcarbonyl groups.

In some embodiments, the organic residue is iminodipropionic acid,iminodiacetic acid, 4-amino-pimelic acid, 4-amino-heptanedioic acid,3-aminohexanedioic acid, 3-aminoadipic acid, 2-aminooctanedioic acid, or2-amino-6-carbonyl-heptanedioic acid.

In some embodiments, the branched residue is a lysine or a lysinederivative, arginine or an arginine derivative, aspartic acid or anaspartic acid derivative, glutamic acid or a glutamic acid derivative,asparagines or a asparagines derivative, glutamine or glutaminederivative, tyrosine or tyrosine derivative, cysteine or cysteinederivative or ornithine or ornithine derivative.

In some embodiments, the branched residue is an amino acid substitutedat the N-position with a residue containing a terminal amino or carbonylreactive group.

In some embodiments, the branched residue is an organic residuecontaining two or more terminal amino groups or two or more terminalcarbonyl groups.

In some embodiments, the branched residue is an organic residuecontaining two or more terminal amino groups. In some embodiments, thebranched residue is an organic residue containing two or more terminalcarbonyl groups. In some embodiments, the branched residue is adiaminopropionic acid. In some embodiments, the branched residue is adiaminopropionic carbonyl compound.

In some embodiments, the branched residue is4-(carbonylmethoxy)phenylalanine, 2-amino-6-(carbonylmethylamino)hexanoic acid, S-(carbonylpropyl)cysteine, S-(carbonylethyl)cysteine,S-(carbonylmethyl)cysteine, N-(carbonylethyl)glycine,N-(carbonylmethyl)glycine, iminodipropionic acid, iminodiacetic acid,4-amino-pimelic acid, 4-amino-heptanedioic acid, 3-aminohexanedioicacid, 3-aminoadipic acid, 2-aminooctanedioic acid, or2-amino-6-carbonyl-heptanedioic acid.

In some embodiments, the branched residue is prepared fromFmoc-L-Asp-AMC, Fmoc-L-Asp-pNA, Fmoc-L-Glu-AMC, Fmoc-L-Glu-pNA,Fmoc-L-Glu(Edans)-OH, Fmoc-L-Glu(PEG-biotinyl)-OH,(S)-Fmoc-2-amino-hexanedioic acid-6-tert-butyl ester,(S)-Fmoc-2-amino-adipic acid-6-tert-butyl ester, (S)-Fmoc-Aad(OtBu)-OH,(S)-Fmoc-2-amino-5-tert-butoxycarbonyl-hexanedioic acid-6-tert-butylester, (S)-Fmoc-2-amino-heptanedioic acid-7-tert-butyl ester,(S)-Fmoc-2-amino-pimelic acid-7-tert-butyl ester,(S)-Fmoc-2-amino-6-tert-butoxycarbonyl-heptanedioic acid—7-tert-butylester, (S)-Fmoc-2-amino-octanedioic acid-8-tert-butyl ester,(S)-Fmoc-2-amino-suberic acid-8-tert-butyl ester, (S)-Fmoc-Asu(OtBu)-OH,(R)-Fmoc-3-amino-hexanedioic acid-1-tert-butyl ester,(R)-Fmoc-3-amino-adipic acid-1-tert-butyl ester,(R)-Fmoc-4-amino-heptanedioic acid-1-tert-butyl ester,(R)-Fmoc-4-amino-pimelic acid-1-tert-butyl ester, Boc-iminodiaceticacid, Fmoc-iminodiacetic acid, Boc-iminodipropionic acid,Fmoc-iminodipropionic acid, Fmoc-N-(tert-butoxycarbonylmethyl)-glycine,Fmoc-N-(tert-butoxycarbonylethyl)-glycine,Fmoc-L-Cys(tert-butoxycarbonylmethyl)-OH(R)-Fmoc-2-amino-3-(tert-butoxycarbonylmethylsulfanyl)-propionic acid,Fmoc-L-Cys(tert-butoxycarbonylpropyl)-OH(R)-Fmoc-2-amino-3-(3-tert-butoxycarbonylpropylsulfanyl)-propionic acid,Fmoc-L-Cys(tert-butoxycarbonylethyl)-OH(R)-Fmoc-2-amino-3-(2-tert-butoxycarbonylethylsulfanyl)-propionic acid,Fmoc-4-(tert-butoxycarbonylmethoxy)-L-phenylalanine, or(S)-Fmoc-2-amino-6-(Boc-tert-butoxycarbonylmethylamino)-hexanoic acid.

In some embodiments, the branched residue is prepared fromN-α-Boc-DL-diaminopropionic acid, N-α-Boc-D-diaminopropionic acid,N-α-Boc-L-diaminopropionic acid, N-α-Fmoc-L-diaminopropionic acid,N-α-Boc-N—S-Alloc-D-diaminopropionic acid,N-α-Boc-N—S-Alloc-L-diaminopropionic acid,N-α-Fmoc-N—S-alloc-L-diaminopropionic acid,N-α-N—S-Bis-Boc-L-diaminopropionic acid,N-α-Fmoc-N—S-Boc-D-diaminopropionic acid,N-α-Fmoc-N—S-Boc-L-diaminopropionic acid,N-α-Z—N—S-Boc-L-diaminopropionic acid,N-α-Boc-N—S-Fmoc-D-diaminopropionic acid,N-α-Boc-N—S-Fmoc-L-diaminopropionic acid,N-α-N—S-Bis-Fmoc-L-diaminopropionic acid,N-α-Z—N—S-Fmoc-L-diaminopropionic acid, N-α-Boc-N—S—Z-L-diaminopropionicacid, N-α-Fmoc-N—S—Z-L-diaminopropionic acid,N-α-Fmoc-N—S-(Boc-aminooxyacetyl)-L-diaminopropionic acid,N-α-Boc-N-gamma-Fmoc-D-diaminobutyric acid,N-α-Boc-N-gamma-Fmoc-L-diaminobutyric acid,N-α-Boc-N-gamma-Fmoc-L-diaminobutyric acid,N-α-Fmoc-N-gamma-Boc-D-diaminobutyric acid,N-α-Fmoc-N-gamma-Boc-L-diaminobutyric acid,N-α-Fmoc-N-gamma-Alloc-L-diaminobutyric acid,(S)—N-b-Fmoc-N-gamma-Boc-3,4-diaminobutyric acid, H-L-ornithine,N-α-Boc-N-delta-Alloc-L-ornithine, N-α-Fmoc-N-delta-Alloc-L-ornithine,N-α-Fmoc-N-delta-Boc-L-ornithine, (S)-Boc-2-amino-5-azido-pentanoicacid.DCHA, (S)-Fmoc-2-amino-5-azido-pentanoic acid,N-α-N-delta-bis-Boc-N-α-N-delta-bis(3-Boc-aminopropyl)-L-ornithine,N-α-Boc-N—S—N-delta-N-delta-tris(3-Boc-aminopropyl)-L-ornithine,Fmoc-L-Lys(Biotin)-OH, Fmoc-L-Lys(Dabcyl)-OH, Fmoc-L-Lys(Boc) (Me)-OH,Fmoc-L-Lys (Boc) (iPr)-OH,(2S,5R)-Fmoc-2-amino-4-(3-Boc-2,2-dimethyl-oxazolidin-5-yl)-butyricacid, (S)-Fmoc-2-amino-6-(Boc-tert-butoxycarbonylmethyl-amino)-hexanoicacid, (S)-Fmoc-2-amino-7-(Boc-amino)-heptanoic acid, Fmoc-L-Arg(Me)(Pbf)-OH, Fmoc-L-Arg(Me)2(Pbf)-OH, Fmoc-L-Arg(Me)2-OH,(S)-Fmoc-3-amino-5-[(N′-Pbf-pyrrolidine-1-carboximidoyl)-amino]-pentanoicacid, Fmoc-L-Homoarg(Et)2-OH, Boc-3-amino-5-(Fmoc-amino)-benzoic acid,3,5-bis[2-(Boc-amino)ethoxy]-benzoic acid,Fmoc-4-[2-(Boc-amino)ethoxy]-L-phenylalanine,N,N-bis(N′-Fmoc-3-aminopropyl)-glycine potassium hemisulfate,N,N-bis(N′-Fmoc-3-aminopropyl)-glycine potassium hemisulfate,Fmoc-N-(2-Boc-aminoethyl)-glycine, Fmoc-N-(3-Boc-aminopropyl)-glycine,Fmoc-N-(4-Boc-aminobutyl)-glycine, (R,S)—N-α-Fmoc-N-a′-Boc-diaminoaceticacid, N,N′-bis-Fmoc-diaminoacetic acid,(S)—N-4-Fmoc-N-8-Boc-diaminooctanoic acid,(R,S)—N-Fmoc-N′-Boc-imidazolidine-2-carboxylic acid,Fmoc-p(NH-Boc)-L-Phe-OH, Boc-p(NH-Fmoc)-L-Phe-OH, orBoc-p(NH—Z)-L-Phe-OH.

Each embodiment disclosed herein is contemplated as being applicable toeach of the other disclosed embodiments. Thus, all combinations of thevarious elements described herein are within the scope of the invention.

It is understood that where a parameter range is provided, all integerswithin that range, and tenths thereof, are also provided by theinvention. For example, “0.2-5 mg/kg/day” is a disclosure of 0.2mg/kg/day, 0.3 mg/kg/day, 0.4 mg/kg/day, 0.5 mg/kg/day, 0.6 mg/kg/dayetc. up to 5.0 mg/kg/day.

Terms

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below.

Peptidyl linkage: the structure

A peptidyl linkage may be a peptide bond.

Stretch of consecutive amino acids: a plurality of amino acids arrangedin a chain, each of which is joined to a preceding amino acid by apeptide bond, excepting that the first amino acid in the chain mayoptionally not be joined to a preceding amino acid. The amino acids ofthe chain may be naturally or non-naturally occurring, or may comprise amixture thereof.

The amino acids, unless otherwise indicated, may be genetically encoded,naturally-occurring but not genetically encoded, or non-naturallyoccurring, and any selection thereof.

N-terminal amino acid residue: the terminal residue of a stretch of twoor more consecutive amono acids having a free α-amino (NH₂) functionalgroup, or a derivative of an α-amino (NH₂) functional group.

N-terminus: the free α-amino (NH₂) group (or derivative thereof) of aN-terminal amino acid residue.

C-terminal amino acid residue: the terminal residue of a stretch of twoor more consecutive amono acids having a free α-carboxyl (COOH)functional group, or a derivative of a α-carboxyl (COOH) functionalgroup.

C-terminus: the free α-carboxyl (COOH) group (or derivative thereof) ofa C-terminal amino acid residue.

A “bond”, unless otherwise specified, or contrary to context, isunderstood to include a covalent bond, a dipole-dipole interaction suchas a hydrogen bond, and intermolecular interactions such as van derWaals forces.

A “Signal Sequence” is a short (3-60 amino acids long) peptide chainthat directs the post-translational transport of a polypeptide.

“Amino acid” as used herein, in one embodiment, means a L or D isomer ofthe genetically encoded amino acids, i.e. isoleucine, alanine, leucine,asparagine, lysine, aspartate, methionine, cysteine, phenylalanine,glutamate, threonine, glutamine, tryptophan, glycine, valine, proline,arginine, serine, histidine, tyrosine, selenocysteine, pyrrolysine andalso includes homocysteine and homoselenocysteine.

Other examples of amino acids include an L or D isomer of taurine, gaba,dopamine, lanthionine, 2-aminoisobutyric acid, dehydroalanine, ornithineand citrulline, as well as non-natural homologues and syntheticallymodified forms thereof including amino acids having alkylene chainsshortened or lengthened by up to two carbon atoms, amino acidscomprising optionally substituted aryl groups, and amino acidscomprising halogenated groups, including halogenated alkyl and arylgroups as well as beta or gamma amino acids, and cyclic analogs.

Due to the presence of ionizable amino and carboxyl groups, the aminoacids in these embodiments may be in the form of acidic or basic salts,or may be in neutral forms. Individual amino acid residues may also bemodified by oxidation or reduction. Other contemplated modificationsinclude hydroxylation of proline and lysine, phosphorylation of hydroxylgroups of seryl or threonyl residues, and methylation of the alpha-aminogroups of lysine, arginine, and histidine side chains.

Covalent derivatives may be prepared by linking particular functionalgroups to the amino acid side chains or at the N- or C-termini.

Compounds comprising amino acids with R-group substitutions are withinthe scope of the invention. It is understood that substituents andsubstitution patterns on the compounds of the instant invention can beselected by one of ordinary skill in the art to provide compounds thatare chemically stable from readily available starting materials.

“Natural amino acid” as used herein means a L or D isomer of thegenetically encoded amino acids, i.e. isoleucine, alanine, leucine,asparagine, lysine, aspartate, methionine, cysteine, phenylalanine,glutamate, threonine, glutamine, tryptophan, glycine, valine, proline,arginine, serine, histidine, tyrosine, selenocysteine, pyrrolysine andhomocysteine and homoselenocysteine.

“Non-natural amino acid” as used herein means a chemically modified L orD isomer of isoleucine, alanine, leucine, asparagine, lysine, aspartate,methionine, cysteine, phenylalanine, glutamate, threonine, glutamine,tryptophan, glycine, valine, proline, arginine, serine, histidine,tyrosine, selenocysteine, pyrrolysine, homocysteine, homoselenocysteine,taurine, gaba, dopamine, lanthionine, 2-aminoisobutyric acid,dehydroalanine, ornithine or citrulline, including cysteine andselenocysteine derivatives having C₃-C₁₀ aliphatic side chains betweenthe alpha carbon and the S or Se. In one embodiment the aliphatic sidechain is an alkylene. In another embodiment, the aliphatic side chain isan alkenylene or alkynylene.

In addition to the stretches of consecutive amino acid sequencesdescribed herein, it is contemplated that variants thereof can beprepared by introducing appropriate nucleotide changes into the encodingDNA, and/or by synthesis of the desired consecutive amino acidsequences. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the stretches ofconsecutive amino acids described herein when expression is the chosenmethod of synthesis (rather than chemical synthesis for example), suchas changing the number or position of glycosylation sites or alteringthe membrane anchoring characteristics.

Variations in the sequences described herein, can be made, for example,using any of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the consecutive amino acid sequence ofinterest that results in a change in the amino acid sequence as comparedwith the native sequence. Optionally the variation is by substitution ofat least one amino acid with any other amino acid in one or more of thedomains. Guidance in determining which amino acid residue may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence with that of homologousknown protein molecules and minimizing the number of amino acid sequencechanges made in regions of high homology. Amino acid substitutions canbe the result of replacing one amino acid with another amino acid havingsimilar structural and/or chemical properties, such as the replacementof a leucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence. It is understood that anyterminal variations are made within the context of the inventiondisclosed herein.

Amino acid sequence variants of the binding partner are prepared withvarious objectives in mind, including increasing the affinity of thebinding partner for its ligand, facilitating the stability, purificationand preparation of the binding partner, modifying its plasma half life,improving therapeutic efficacy, and lessening the severity or occurrenceof side effects during therapeutic use of the binding partner.

Amino acid sequence variants of these sequences are also contemplatedherein including insertional, substitutional, or deletional variants.Such variants ordinarily can prepared by site-specific mutagenesis ofnucleotides in the DNA encoding the target-binding monomer, by which DNAencoding the variant is obtained, and thereafter expressing the DNA inrecombinant cell culture. Fragments having up to about 100-150 aminoacid residues can also be prepared conveniently by in vitro synthesis.Such amino acid sequence variants are predetermined variants and are notfound in nature. The variants exhibit the qualitative biologicalactivity (including target-binding) of the nonvariant form, though notnecessarily of the same quantative value. While the site for introducingan amino acid sequence variation is predetermined, the mutation per seneed not be predetermined. For example, in order to optimize theperformance of a mutation at a given site, random or saturationmutagenesis (where all 20 possible residues are inserted) is conductedat the target codon and the expressed variant is screened for theoptimal combination of desired activities. Such screening is within theordinary skill in the art.

Amino acid insertions usually will be on the order of about from 1 to 10amino acid residues; substitutions are typically introduced for singleresidues; and deletions will range about from 1 to 30 residues.Deletions or insertions preferably are made in adjacent pairs, i.e. adeletion of 2 residues or insertion of 2 residues. It will be amplyapparent from the following discussion that substitutions, deletions,insertions or any combination thereof are introduced or combined toarrive at a final construct.

In an aspect, the invention concerns a compound comprising a stretch ofconsecutive amino acids having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to an amino acidsequence disclosed in the specification, a figure, a SEQ ID NO. or asequence listing of the present application.

The % amino acid sequence identity values can be readily obtained using,for example, the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460-480 (1996)).

Fragments of native sequences are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Again, it is understood that any terminal variations are made within thecontext of the invention disclosed herein.

Certain fragments lack amino acid residues that are not essential for adesired biological activity of the sequence of interest.

Any of a number of conventional techniques may be used. Desired peptidefragments or fragments of stretches of consecutive amino acids may bechemically synthesized. An alternative approach involves generatingfragments by enzymatic digestion, e.g. by treating the protein with anenzyme known to cleave proteins at sites defined by particular aminoacid residues, or by digesting the DNA with suitable restriction enzymesand isolating the desired fragment. Yet another suitable techniqueinvolves isolating and amplifying a DNA fragment encoding a desiredpolypeptide/sequence fragment, by polymerase chain reaction (PCR).Oligonucleotides that define the desired termini of the DNA fragment areemployed at the 5′ and 3′ primers in the PCR.

In particular embodiments, conservative substitutions of interest areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 1, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 1 Original Exemplary Preferred Ala (A) val; leu; ile val Arg (R)lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C)ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H)asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leuLeu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asnarg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro(P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y)trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu

Substantial modifications in function or immunological identity of thesequence are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;(2) neutral hydrophilic: cys, ser, thr;(3) acidic: asp, glu;(4) basic: asn, gln, his, lys, arg;(5) residues that influence chain orientation: gly, pro;(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)),restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)) or other known techniques can be performedon the cloned DNA to produce the variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant (Cunningham and Wells,Science, 244:1081-1085 (1989)). Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions (Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Covalent modifications: The stretch of consecutive amino acids may becovalently modified. One type of covalent modification includes reactingtargeted amino acid residues with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues that are not involved in an -x-x- bond. Derivatization withbifunctional agents is useful, for instance, for crosslinking to awater-insoluble support matrix or surface for use in the method forpurifying anti-sequence of interest antibodies, and vice-versa. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-((p-azidophenyl)dithio)propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the.alpha.-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman& Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification comprises altering the nativeglycosylation pattern of the stretch of consecutive amino acids.“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found in aminoacid sequences (either by removing the underlying glycosylation site orby deleting the glycosylation by chemical and/or enzymatic means),and/or adding one or more glycosylation sites that are not present inthe native sequence. In addition, the phrase includes qualitativechanges in the glycosylation of the native proteins, involving a changein the nature and proportions of the various carbohydrate moietiespresent.

Addition of glycosylation sites to the amino acid sequence may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence (for O-linkedglycosylation sites). The amino acid sequence may optionally be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the amino acid sequence at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theamino acid sequence is by chemical or enzymatic coupling of glycosidesto the polypeptide. Such methods are described in the art, e.g., in WO87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the amino acid sequence maybe accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification comprises linking the amino acidsequence to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The term “substitution”, “substituted” and “substituent” refers to afunctional group as described above in which one or more bonds to ahydrogen atom contained therein are replaced by a bond to non-hydrogenor non-carbon atoms, provided that normal valencies are maintained andthat the substitution results in a stable compound. Substituted groupsalso include groups in which one or more bonds to a carbon(s) orhydrogen(s) atom are replaced by one or more bonds, including double ortriple bonds, to a heteroatom. Examples of substituent groups includethe functional groups described above, and halogens (i.e., F, Cl, Br,and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl,n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, suchas methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such asphenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) andp-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy);heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl,methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto;sulfanyl groups, such as methylsulfanyl, ethylsulfanyl andpropylsulfanyl; cyano; amino groups, such as amino, methylamino,dimethylamino, ethylamino, and diethylamino; and carboxyl. Wheremultiple substituent moieties are disclosed or claimed, the substitutedcompound can be independently substituted by one or more of thedisclosed or claimed substituent moieties, singly or plurally. Byindependently substituted, it is meant that the (two or more)substituents can be the same or different.

In the compounds used in the method of the present invention, alkyl,heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groupscan be further substituted by replacing one or more hydrogen atoms withalternative non-hydrogen groups. These include, but are not limited to,halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

It is understood that substituents and substitution patterns on thecompounds used in the method of the present invention can be selected byone of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art from readily available starting materials. If asubstituent is itself substituted with more than one group, it isunderstood that these multiple groups may be on the same carbon or ondifferent carbons, so long as a stable structure results.

In choosing the compounds used in the method of the present invention,one of ordinary skill in the art will recognize that the varioussubstituents, i.e. R₁, R₂, etc. are to be chosen in conformity withwell-known principles of chemical structure connectivity.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms and may be unsubstituted or substituted. Thus, C₁-C_(n) asin “C₁-C_(n) alkyl” is defined to include groups having 1, 2, . . . ,n−1 or n carbons in a linear or branched arrangement. For example,C₁-C₆, as in “C₁-C₆ alkyl” is defined to include groups having 1, 2, 3,4, 5, or 6 carbons in a linear or branched arrangement, and specificallyincludes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl,and hexyl. Unless otherwise specified contains one to ten carbons. Alkylgroups can be unsubstituted or substituted with one or moresubstituents, including but not limited to halogen, alkoxy, alkylthio,trifluoromethyl, difluoromethyl, methoxy, and hydroxyl.

As used herein, “C₁-C₄ alkyl” includes both branched and straight-chainC₁-C₄ alkyl.

As used herein, “aryl” is intended to mean any stable monocyclic,bicyclic or polycyclic carbon ring of up to 10 atoms in each ring,wherein at least one ring is aromatic, and may be unsubstituted orsubstituted. Examples of such aryl elements include but are not limitedto: phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl,indanyl, phenanthryl, anthryl or acenaphthyl. In cases where the arylsubstituent is bicyclic and one ring is non-aromatic, it is understoodthat attachment is via the aromatic ring.

The term “phenyl” is intended to mean an aromatic six membered ringcontaining six carbons, and any substituted derivative thereof.

The term “benzyl” is intended to mean a methylene attached directly to abenzene ring. A benzyl group is a methyl group wherein a hydrogen isreplaced with a phenyl group, and any substituted derivative thereof.

The compounds used in the method of the present invention may beprepared by techniques well know in organic synthesis and familiar to apractitioner ordinarily skilled in the art. However, these may not bethe only means by which to synthesize or obtain the desired compounds.

The compounds of present invention may be prepared by techniquesdescribed in Vogel's Textbook of Practical Organic Chemistry, A. I.Vogel, A. R. Tatchell, B. S. Furnis, A. J. Hannaford, P. W. G. Smith,(Prentice Hall) 5^(th) Edition (1996), March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, JerryMarch, (Wiley-Interscience) 5^(th) Edition (2007), and referencestherein, which are incorporated by reference herein. However, these maynot be the only means by which to synthesize or obtain the desiredcompounds.

In some embodiments of the present invention, a compound comprises anonproteinaceous polymer. In some embodiments, the nonproteinaceouspolymer may be is a hydrophilic synthetic polymer, i.e., a polymer nototherwise found in nature. However, polymers which exist in nature andare produced by recombinant or in vitro methods are useful, as arepolymers which are isolated from nature. Hydrophilic polyvinyl polymersfall within the scope of this invention, e.g. polyvinylalcohol andpolyvinylpyrrolidone. Particularly useful are polyalkylene ethers suchas polyethylene glycol, polypropylene glycol, polyoxyethylene esters ormethoxy polyethylene glycol; polyoxyalkylenes such as polyoxyethylene,polyoxypropylene, and block copolymers of polyoxyethylene andpolyoxypropylene (Pluronics);

polymethacrylates; carbomers; branched or unbranched polysaccharideswhich comprise the saccharide monomers D-mannose, D- and L-galactose,fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,D-galacturontc acid, D-mannuronic acid (e.g. polymannuronic acid, oralginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminicacid including homopolysaccharides and heteropolysaccharides such aslactose, amylopectin, starch, hydroxyethyl starch, amylose, dextransulfate, dextran, dextrins, glycogen, or the polysaccharide subunit ofacid mucopolysaccharides,e.g. hyaluronic acid; polymers of sugar alcohols such as polysorbitoland polymannitol; and heparin or heparon.

Salts

Salts of the compounds disclosed herein are within the scope of theinvention. As used herein, a “salt” is salt of the instant compoundswhich has been modified by making acid or base salts of the compounds.

Fc Domains

The term “Fc domain”, as used herein, generally refers to a monomer ordimer complex, comprising the C-terminal polypeptide sequences of animmunoglobulin heavy chain. The Fc domain may comprise native or variantFc sequences. Although the boundaries of the Fc domain of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcdomain is usually defined to stretch from an amino acid residue in thehinge region to the carboxyl terminus of the Fc sequence. The Fcsequence of an immunoglobulin generally comprises two constant regions,a CH2 region and a CH3 region, and optionally comprises a CH4 region. Ahuman Fc domain may be obtained from any suitable immunoglobulin, suchas the IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM.

Suitable Fc domains are prepared by recombinant DNA expression of pre-Fcchimeric polypeptides comprising 1) a signal peptide, obtained from asecreted or transmembrane protein, that is cleaved in front of a maturepolypeptide having an N-terminal cysteine residue, contiguous with 2) anFc domain polypeptide having an N-terminal cysteine residue.

Suitable examples of signal peptides are sonic hedgehog (SHH) (GenBankAcc. No. NM000193), IFNalpha-2 (IFN) (GenBank Acc. No. NP000596), andcholesterol ester transferase (CETP) (GenBank Accession No. NM000078).Other suitable examples include Indian hedgehog (Genbank Acc. No.NM002181), desert hedgehog (Genbank Acc. No. NM021044), IFNalpha-1(Genbank Acc. No. NP076918), IFNalpha-4 (Genbank Acc. No. NM021068),IFNalpha-5 (Genbank Acc. No. NM002169), IFNalpha-6 (Genbank Acc. No.NM021002), IFNalpha-7 (Genbank Acc. No. NM021057), IFNalpha-8 (GenbankAcc. No. NM002170), IFNalpha-10 (Genbank Acc. No. NM002171), IFNalpha-13(Genbank Acc. No. NM006900), IFNalpha-14 (Genbank Acc. No. NM002172),IFNalpha-16 (Genbank Acc. No. NM002173), IFNalpha-17 (Genbank Acc. No.NM021268) and IFNalpha-21 (Genbank Acc. No. NM002175).

Suitable examples of Fc domains and their pre-Fc chimeric polypeptidesare shown in SEQ ID NO: 1 through SEQ ID NO: 96. The Fc domains areobtained by expressing the pre-Fc chimeric polypeptides in cells underconditions leading to their secretion and cleavage of the signalpeptide. The pre-Fc polypeptides may be expressed in either prokaryoticor eukaryotic host cells. Preferably, mammalian host cells aretransfected with expression vectors encoding the pre-Fc polypeptides.

Human IgG1 Fc domains having the N-terminal sequence CDKTHTCPPCPAPE,CPPCPAPE, and CPAPE are shown in SEQ ID NO: 1, SEQ ID NO: 9, and SEQ IDNO: 17, respectively, and the DNA sequences encoding them are shown inSEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 18, respectively. The IgG1domain of SEQ ID NO: 1 is obtained by expressing the pre-Fc chimericpolypeptides shown in SEQ ID NO: 3 (SHH signal peptide), SEQ ID NO: 5(IFN signal peptide), and SEQ ID NO: 7 (CETP signal peptide), using theDNA sequences shown in SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8,respectively. The IgG1 domain of SEQ ID NO: 9 is obtained by expressingthe pre-Fc chimeric polypeptides shown in SEQ ID NO: 11 (SHH signalpeptide), SEQ ID NO: 13 (IFN signal peptide), and SEQ ID NO: 15 (CETPsignal peptide), using the DNA sequences shown in SEQ ID NO: 12, SEQ IDNO: 14, and SEQ ID NO: 16, respectively. The IgG1 domain of SEQ ID NO:17 is obtained by expressing the pre-Fc chimeric polypeptides shown inSEQ ID NO: 19 (SHH signal peptide), SEQ ID NO: 21 (IFN signal peptide),and SEQ ID NO: 23 (CETP signal peptide), using the DNA sequences shownin SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24, respectively.

Human IgG2 Fc domains having the N-terminal sequence CCVECPPCPAPE,CVECPPCPAPE, CPPCPAPE, and CPAPE are shown in SEQ ID NO: 25, SEQ ID NO:33, SEQ ID NO: 41, and SEQ ID NO: 49, respectively, and the DNAsequences encoding them are shown in SEQ ID NO: 26, SEQ ID NO: 34, SEQID NO: 42, and SEQ ID NO: 50, respectively. The IgG2 domain of SEQ IDNO: 25 is obtained by expressing the pre-Fc chimeric polypeptides shownin SEQ ID NO: 27 (SHH signal peptide), SEQ ID NO: 29 (IFN signalpeptide), and SEQ ID NO: 31 (CETP signal peptide), using the DNAsequences shown in SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32,respectively. The IgG2 domain of SEQ ID NO: 33 is obtained by expressingthe pre-Fc chimeric polypeptides shown in SEQ ID NO: 35 (SHH signalpeptide), SEQ ID NO: 37 (IFN signal peptide), and SEQ ID NO: 39 (CETPsignal peptide) using the DNA sequences shown in SEQ ID NO: 36, SEQ IDNO: 38, and SEQ ID NO: 40, respectively. The IgG2 domain of SEQ ID NO:41 is obtained from the pre-Fc chimeric polypeptides shown in SEQ ID NO:43 (SHH signal peptide), SEQ ID NO: 45 (IFN signal peptide), and SEQ IDNO: 47 (CETP signal peptide), using the DNA sequences shown in SEQ IDNO: 44, SEQ ID NO: 46, and SEQ ID NO: 48, respectively. The IgG2 domainof SEQ ID NO: 49 is obtained from the pre-Fc chimeric polypeptides shownin SEQ ID NO: 51 (SHH signal peptide), SEQ ID NO: 53 (IFN signalpeptide), and SEQ ID NO: 55 (CETP signal peptide), using the DNAsequences shown in SEQ ID NO: 52, SEQ ID NO: 54, and SEQ ID NO: 56,respectively.

Human IgG3 Fc domains having the N-terminal sequence(CPRCPEPKSDTPPP)₃-CPRCPAPE, CPRCPAPE, and CPAPE are shown in SEQ ID NO:57, SEQ ID NO: 65, and SEQ ID NO: 73, respectively, and the DNAsequences encoding them are shown in SEQ ID NO: 58, SEQ ID NO: 66, SEQID NO: 42, and SEQ ID NO: 74, respectively. The IgG3 domain of SEQ IDNO: 57 is obtained by expressing the pre-Fc chimeric polypeptides shownin SEQ ID NO: 59 (SHH signal peptide), SEQ ID NO: 61 (IFN signalpeptide), and SEQ ID NO: 63 (CETP signal peptide), using the DNAsequences shown in SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64,respectively. The IgG3 domain of SEQ ID NO: 65 is obtained by expressingthe pre-Fc chimeric polypeptides shown in SEQ ID NO: 67 (SHH signalpeptide), SEQ ID NO: 69 (IFN signal peptide), and SEQ ID NO: 71 (CETPsignal peptide), using the DNA sequences shown in SEQ ID NO: 68, SEQ IDNO: 70, and SEQ ID NO: 72, respectively. The IgG3 domain of SEQ ID NO:73 is obtained by expressing the pre-Fc chimeric polypeptides shown inSEQ ID NO: 75 (SHH signal peptide), SEQ ID NO: 77 (IFN signal peptide),and SEQ ID NO: 79 (CETP signal peptide), using the DNA sequences shownin SEQ ID NO: 76, SEQ ID NO: 78, and SEQ ID NO: 80, respectively.

The sequences of human IgG4 Fc domains having the N-terminal sequenceCPSCPAPE and CPAPE are shown in SEQ ID NO: 81 and SEQ ID NO: 89,respectively, and the DNA sequences encoding them are shown in SEQ IDNO: 82 and SEQ ID NO: 90, respectively. The IgG4 domain of SEQ ID NO: 81is obtained by expressing the pre-Fc chimeric polypeptides shown in SEQID NO: 83 (SHH signal peptide), SEQ ID NO: 85 (IFN signal peptide), andSEQ ID NO: 87 (CETP signal peptide), using the DNA sequences shown inSEQ ID NO: 84, SEQ ID NO: 86, and SEQ ID NO: 88, respectively. The IgG4domain of SEQ ID NO: 89 is obtained by expressing the pre-Fc chimericpolypeptides shown in SEQ ID NO: 91 (SHH signal peptide), SEQ ID NO: 93(IFN signal peptide), and SEQ ID NO: 95 (CETP signal peptide), using theDNA sequences shown in SEQ ID NO: 92, SEQ ID NO: 94, and SEQ ID NO: 96,respectively.

Suitable host cells include 293 human embryonic cells (ATCC CRL-1573)and CHO-K1 hamster ovary cells (ATCC CCL-61) obtained from the AmericanType Culture Collection (Rockville, Md.). Cells are grown at 37.degree.C. in an atmosphere of air, 95%; carbon dioxide, 5%. 293 cells aremaintained in Minimal essential medium (Eagle) with 2 mM L-glutamine andEarle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mMnon-essential amino acids, and 1.0 mM sodium pyruvate, 90%; fetal bovineserum, 10%. CHO-K1 cells are maintained in Ham's F12K medium with 2 mML-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 90%; fetalbovine serum, 10%. Other suitable host cells include CV1 monkey kidneycells (ATCC CCL-70), COS-7 monkey kidney cells (ATCC CRL-1651), VERO-76monkey kidney cells (ATCC CRL-1587), HELA human cervical cells (ATCCCCL-2), W138 human lung cells (ATCC CCL-75), MDCK canine kidney cells(ATCC CCL-34), BRL3A rat liver cells (ATCC CRL-1442), BHK hamster kidneycells (ATCC CCL-10), MMT060562 mouse mammary cells (ATCC CCL-51), andhuman CD8.sup.+ T lymphocytes (described in U.S. Ser. No. 08/258,152incorporated herein in its entirety by reference).

Examples of a suitable expression vectors are pCDNA3.1(+) shown in SEQID NO: 97 and pSA shown in SEQ ID NO: 98. Plasmid pSA contains thefollowing DNA sequence elements: 1) pBluescriptIIKS(+) (nucleotides912-2941/1-619, GenBank Accession No. X52327), 2) a humancytomegalovirus promoter, enhancer, and first exon splice donor(nucleotides 63-912, GenBank Accession No. K03104), 3) a humanalpha1-globin second exon splice acceptor (nucleotides 6808-6919,GenBank Accession No. J00153), 4) an SV40 T antigen polyadenylation site(nucleotides 2770-2533, Reddy et al. (1978) Science 200, 494-502), and5) an SV40 origin of replication (nucleotides 5725-5578, Reddy et al.,ibid). Other suitable expression vectors include plasmids pSVeCD4DHFRand pRKCD4 (U.S. Pat. No. 5,336,603), plasmid pIK.1.1 (U.S. Pat. No.5,359,046), plasmid pVL-2 (U.S. Pat. No. 5,838,464), plasmid pRT43.2F3(described in U.S. Ser. No. 08/258,152 incorporated herein in itsentirety by reference).

Suitable expression vectors for human IgG pre-Fc polypeptides may beconstructed by the ligation of a HindIII-PspOM1 vector fragment preparedfrom SEQ ID NO: 98, with a HindIII-EagI insert fragment prepared fromSEQ ID NOS: 4, 6, 8, 12, 14, 16, 20, 22, 24, 28, 30, 32, 36, 38, 40, 44,46, 48, 52, 54, 56, 60, 62, 64, 68, 70, 72, 76, 78, 80, 84, 86, 88, 92,94, and 96.

Suitable selectable markers include the Tn5 transposon neomycinphosphotransferase (NEO) gene (Southern and Berg (1982) J. Mol. Appl.Gen. 1, 327-341), and the dihydrofolate reductase (DHFR) cDNA (Lucas etal. (1996) Nucl. Acids Res. 24, 1774-1779). One example of a suitableexpression vector that incorporates a NEO gene is plasmid pSA-NEO, whichis constructed by ligating a first DNA fragment, prepared by digestingSEQ ID NO: 99 with EcoRI and BglII, with a second DNA fragment, preparedby digesting SEQ ID NO:98 with EcoRI and BglII. SEQ ID NO:99incorporates a NEO gene (nucleotides 1551 to 2345, Genbank Accession No.U00004) preceded by a sequence for translational initiation (Kozak(1991) J. Biol. Chem, 266, 19867-19870). Another example of a suitableexpression vector that incorporates a NEO gene and a DHFR cDNA isplasmid pSVe-NEO-DHFR, which is constructed by ligating a first DNAfragment, prepared by digesting SEQ ID NO:99 with EcoRI and BglII, witha second DNA fragment, prepared by digesting pSVeCD4DHFR with EcoRI andBglII. Plasmid pSVe-NEO-DHFR uses SV40 early promoter/enhancers to driveexpression of the NEO gene and the DHFR cDNA. Other suitable selectablemarkers include the XPGT gene (Mulligan and Berg (1980) Science 209,1422-1427) and the hygromycin resistance gene (Sugden et al. (1985) Mol.Cell. Biol. 5, 410-413).

In one embodiment, cells are transfected by the calcium phosphate methodof Graham et al. (1977) J. Gen. Virol. 36, 59-74. A DNA mixture (10 ug)is dissolved in 0.5 ml of 1 mM Tris-HCl, 0.1 mM EDTA, and 227 mM CaCl₂.The DNA mixture contains (in a ratio of 10:1:1) the expression vectorDNA, the selectable marker DNA, and a DNA encoding the VA RNA gene(Thimmappaya et al. (1982) Cell 31, 543-551). To this mixture is added,dropwise, 0.5 mL of 50 mM Hepes (pH 7.35), 280 mM NaCl, and 1.5 mMNaPO₄. The DNA precipitate is allowed to form for 10 minutes at 25° C.,then suspended and added to cells grown to confluence on 100 mm plastictissue culture dishes. After 4 hours at 37° C., the culture medium isaspirated and 2 ml of 20% glycerol in PBS is added for 0.5 minutes. Thecells are then washed with serum-free medium, fresh culture medium isadded, and the cells are incubated for 5 days.

In another embodiment, cells are transiently transfected by the dextransulfate method of Somparyrac et al. (1981) Proc. Nat. Acad. Sci. 12,7575-7579. Cells are grown to maximal density in spinner flasks,concentrated by centrifugation, and washed with PBS. The DNA-dextranprecipitate is incubated on the cell pellet. After 4 hours at 37° C.,the DEAE-dextran is aspirated and 20% glycerol in PBS is added for 1.5minutes. The cells are then washed with serum-free medium, re-introducedinto spinner flasks containing fresh culture medium with 5 micrograms/mlbovine insulin and 0.1 micrograms/ml bovine transferring, and incubatedfor 4 days.

Following transfection by either method, the conditioned media iscentrifuged and filtered to remove the host cells and debris. The samplecontained the Fc domain is then concentrated and purified by anyselected method, such as dialysis and/or column chromatography (seebelow). To identify the Fc domain in the cell culture supernatant, theculture medium is removed 24 to 96 hours after transfection,concentrated, and analyzed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) in the presence or absence of a reducing agent such asdithiothreitol.

For unamplified expression, plasmids are transfected into human 293cells (Graham et al., J. Gen. Virol. 36:59 74 (1977)), using a highefficiency procedure (Gorman et al., DNA Prot. Eng. Tech. 2:3 10(1990)). Media is changed to serum-free and harvested daily for up tofive days. For unamplified expression, plasmids are transfected intohuman 293 cells (Graham et al., J. Gen. Virol. 36:59 74 (1977)), using ahigh efficiency procedure (Gorman et al., DNA Prot. Eng. Tech. 2:3 10(1990)). Media is changed to serum-free and harvested daily for up tofive days. The Fc domains are purified from the cell culture supernatantusing HiTrap Protein A HP (Pharmacia). The eluted Fc domains arebuffer-exchanged into PBS using a Centricon-30 (Amicon), concentrated to0.5 ml, sterile filtered using a Millex-GV (Millipore) at 4° C.

Stretches of Consecutive Amino Acids

Examples of stretches of consecutive amino acids as referred to hereininclude, but are not limited to, consecutive amino acids includingbinding domains such as secreted or transmembrane proteins,intracellular binding domains and antibodies (whole or portions thereof)and modified versions thereof. The following are some non-limitingexamples:

1) Immunoglobulins

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), monovalent antibodies, multivalent antibodies,and antibody fragments so long as they exhibit the desired biologicalactivity (e.g., Fab and/or single-armed antibodies).

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called a, 6,e, y, and p, respectively.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds.

Examples of antibody fragments include but are not limited to Fv, Fab,Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chainantibody molecules (e.g., scFv); and multispecific antibodies formedfrom antibody fragments.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “blocking” antibody or an “antagonist” antibody is one whichsignificantly inhibits (either partially or completely) a biologicalactivity of the antigen it binds.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The phrase “N-terminally truncated heavy chain”, as used herein, refersto a polypeptide comprising parts but not all of a full lengthimmunoglobulin heavy chain, wherein the missing parts are those normallylocated on the N terminal region of the heavy chain. Missing parts mayinclude, but are not limited to, the variable domain, CH1, and part orall of a hinge sequence. Generally, if the wild type hinge sequence isnot present, the remaining constant domain(s) in the N-terminallytruncated heavy chain would comprise a component that is capable oflinkage to another Fc sequence (i.e., the “first” Fc polypeptide asdescribed herein). For example, said component can be a modified residueor an added cysteine residue capable of forming a disulfide linkage.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see, e.g., Daeron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) and regulation of homeostasis ofimmunoglobulins. Methods of measuring binding to FcRn are known (see,e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie etal., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol.Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

Binding to human FcRn in vivo and serum half life of human FcRn highaffinity binding polypeptides can be assayed, e.g., in transgenic miceor transfected human cell lines expressing human FcRn, or in primates towhich the polypeptides with a variant Fc region are administered. WO2000/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. See also, e.g., Shields et al. J. Biol.Chem. 9(2):6591-6604 (2001).

The “hinge region,” “hinge sequence”, and variations thereof, as usedherein, includes the meaning known in the art, which is illustrated in,for example, Janeway et al., Immuno Biology: the immune system in healthand disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al.,Protein Science (1997), 6:407-415; Humphreys et al., J. Immunol. Methods(1997), 209:193-202.

Unless indicated otherwise, the expression “multivalent antibody” isused throughout this specification to denote an antibody comprisingthree or more antigen binding sites. The multivalent antibody ispreferably engineered to have the three or more antigen binding sitesand is generally not a native sequence IgM or IgA antibody.

An “Fv” fragment is an antibody fragment which contains a completeantigen recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in tight association,which can be covalent in nature, for example in scFv. It is in thisconfiguration that the three HVRs of each variable domain interact todefine an antigen binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six HVRs or a subset thereof confer antigen bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three HVRs specific for an antigen) hasthe ability to recognize and bind antigen, although usually at a loweraffinity than the entire binding site.

The “Fab” fragment contains a variable and constant domain of the lightchain and a variable domain and the first constant domain (CH1) of theheavy chain. F(ab′) 2 antibody fragments comprise a pair of Fabfragments which are generally covalently linked near their carboxytermini by hinge cysteines between them. Other chemical couplings ofantibody fragments are also known in the art.

The phrase “antigen binding arm”, as used herein, refers to a componentpart of an antibody fragment that has an ability to specifically bind atarget molecule of interest. Generally and preferably, the antigenbinding arm is a complex of immunoglobulin polypeptide sequences, e.g.,HVR and/or variable domain sequences of an immunoglobulin light andheavy chain.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains, which enablesthe scFv to form the desired structure for antigen binding. For a reviewof scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) and V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The expression “linear antibodies” refers to the antibodies described inZapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V.sub.H—C.sub.H1-V.sub.H—C.sub.H1) which, together with complementarylight chain polypeptides, form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used may be made by a variety of techniques,including but not limited to the hybridoma method, recombinant DNAmethods, phage-display methods, and methods utilizing transgenic animalscontaining all or part of the human immunoglobulin loci, such methodsand other exemplary methods for making monoclonal antibodies beingdescribed herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 Daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more HVRs, compared to a parent antibody whichdoes not possess such alterations, such alterations resulting in animprovement in the affinity of the antibody for antigen.

An antibody having a “biological characteristic” of a designatedantibody is one which possesses one or more of the biologicalcharacteristics of that antibody which distinguish it from otherantibodies that bind to the same antigen.

A “functional antigen binding site” of an antibody is one which iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same. For the multimericantibodies herein, the number of functional antigen binding sites can beevaluated using ultracentrifugation analysis as described in Example 2of U.S. Patent Application Publication No. 20050186208. According tothis method of analysis, different ratios of target antigen tomultimeric antibody are combined and the average molecular weight of thecomplexes is calculated assuming differing numbers of functional bindingsites. These theoretical values are compared to the actual experimentalvalues obtained in order to evaluate the number of functional bindingsites.

A “species-dependent antibody” is one which has a stronger bindingaffinity for an antigen from a first mammalian species than it has for ahomologue of that antigen from a second mammalian species. Normally, thespecies-dependent antibody “binds specifically” to a human antigen (i.e.has a binding affinity (K.sub.d) value of no more than about1.times.10.sup.-7 M, preferably no more than about 1.times.10.sup.-8 Mand most preferably no more than about 1.times.10.sup.-9 M) but has abinding affinity for a homologue of the antigen from a second nonhumanmammalian species which is at least about 50 fold, or at least about 500fold, or at least about 1000 fold, weaker than its binding affinity forthe human antigen. The species-dependent antibody can be any of thevarious types of antibodies as defined above. In some embodiments, thespecies-dependent antibody is a humanized or human antibody.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

2) Extracellular Proteins

Extracellular proteins play important roles in, among other things, theformation, differentiation and maintenance of multicellular organisms. Adiscussion of various intracellular proteins of interest is set forth inU.S. Pat. No. 6,723,535, Ashkenazi et al., issued Apr. 20, 2004, herebyincorporated by reference.

The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, including aspharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature (see, for example, Klein etal., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No.5,536,637)).

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interactions. The membrane-boundproteins can also be employed for screening of potential peptide orsmall molecule inhibitors of the relevant receptor/ligand interaction.

3) Intein-Based C-Terminal Syntheses

As described, for example, in U.S. Pat. No. 6,849,428, issued Feb. 1,2005, inteins are the protein equivalent of the self-splicing RNAintrons (see Perler et al., Nucleic Acids Res. 22:1125-1127 (1994)),which catalyze their own excision from a precursor protein with theconcomitant fusion of the flanking protein sequences, known as exteins(reviewed in Perler et al., Curr. Opin. Chem. Biol. 1:292-299 (1997);Perler, F. B. Cell 92(1):1-4 (1998); Xu et al., EMBO J. 15(19):5146-5153(1996)).

Studies into the mechanism of intein splicing led to the development ofa protein purification system that utilized thiol-induced cleavage ofthe peptide bond at the N-terminus of the Sce VMA intein (Chong et al.,Gene 192(2):271-281 (1997)). Purification with this intein-mediatedsystem generates a bacterially-expressed protein with a C-terminalthioester (Chong et al., (1997)). In one application, where it isdescribed to isolate a cytotoxic protein, the bacterially expressedprotein with the C-terminal thioester is then fused to achemically-synthesized peptide with an N-terminal cysteine using thechemistry described for “native chemical ligation” (Evans et al.,Protein Sci. 7:2256-2264 (1998); Muir et al., Proc. Natl. Acad. Sci. USA95:6705-6710 (1998)).

This technique, referred to as “intein-mediated protein ligation” (IPL),represents an important advance in protein semi-synthetic techniques.However, because chemically-synthesized peptides of larger than about100 residues are difficult to obtain, the general application of IPL waslimited by the requirement of a chemically-synthesized peptide as aligation partner.

IPL technology was significantly expanded when an expressed protein witha predetermined N-terminus, such as cysteine, was generated, asdescribed for example in U.S. Pat. No. 6,849,428. This allows the fusionof one or more expressed proteins from a host cell, such as bacterial,yeast or mammalian cells. In one non-limiting example the intein amodified RIR1 Methanobacterium thermoautotrophicum is that cleaves ateither the C-terminus or N-terminus is used which allows for the releaseof a bacterially expressed protein during a one-column purification,thus eliminating the need proteases entirely.

Intein technology is one example of one route to obtain components. Inone embodiment, the subunits of the compounds of the invention areobtained by transfecting suitable cells, capable of expressing andsecreting mature chimeric polypeptides, wherein such polypeptidescomprise, for example, an adhesin domain contiguous with an isolatablec-terminal intein domain (see U.S. Pat. No. 6,849,428, Evans et al.,issued Feb. 1, 2005, hereby incorporated by reference).

The cells, such as mammalian cells or bacterial cells, are transfectedusing known recombinant DNA techniques. The secreted chimericpolypeptide can then be isolated, e.g. using a chitin-derivatized resinin the case of an intein-chitin binding domain (see U.S. Pat. No.6,897,285, Xu et al., issued May 24, 2005, hereby incorporated byreference), and is then treated under conditions permittingthiol-mediated cleavage and release of the now C-terminalthioester-terminated subunit. The thioester-terminated adhesion subunitis readily converted to a C-terminal cysteine terminated subunit.

For example, following an intein autocleavage reaction, a thioesterintermediate is generated that permits the facile addition of cysteine,selenocysteine, homocysteine, or homoselenocysteine, or a derivative ofcysteine, selenocysteine, homocysteine, homoselenocysteine, to theC-terminus by native chemical ligation. Methods of adding a cysteine,selenocysteine, homocysteine, or homoselenocysteine, or a derivative ofcysteine, selenocysteine, homocysteine, homoselenocysteine, to theC-terminus by native chemical ligation which are useful in aspects ofthe present invention are described in U.S. Patent Application No.2008/0254512, Capon, published Oct. 16, 2008, the entire contents ofwhich are hereby incorporated herein by reference.

Kits

Another aspect of the present invention provides kits comprising thecompounds disclosed herein and the pharmaceutical compositionscomprising these compounds. A kit may include, in addition to thecompound or pharmaceutical composition, diagnostic or therapeuticagents. A kit may also include instructions for use in a diagnostic ortherapeutic method. In a diagnostic embodiment, the kit includes thecompound or a pharmaceutical composition thereof and a diagnostic agent.In a therapeutic embodiment, the kit includes the antibody or apharmaceutical composition thereof and one or more therapeutic agents,such as an additional antineoplastic agent, anti-tumor agent orchemotherapeutic agent.

General Techniques

The description below relates primarily to production of stretches ofconsecutive amino acids or polypeptides of interest by culturing cellstransformed or transfected with a vector containing an encoding nucleicacid. It is, of course, contemplated that alternative methods, which arewell known in the art, may be employed. For instance, the amino acidsequence, or portions thereof, may be produced by direct peptidesynthesis using solid-phase techniques (see, e.g., Stewart et al.,Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif.(1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)). In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of the stretches ofconsecutive amino acids or polypeptides of interest may be chemicallysynthesized separately and combined using chemical or enzymatic methodsto produce the full-length stretches of consecutive amino acids orpolypeptides of interest.

1. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH and the like, canbe selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946(1977) and Hsiao et al.,Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods forintroducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations, e.g., polybrene, polyornithine, may also be used. Forvarious techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature,336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonAptr3phoA E15 (argF-lac)169degP ompT kan.sup.r; E. coli W3110 strain 37D6, which has the completegenotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan.sup.r,E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycinresistant degP deletion mutation; and an E. coli strain having mutantperiplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued Aug. 7,1990. Alternatively, in vitro methods of cloning, e.g., PCR or othernucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for encodingvectors. Saccharomyces cerevisiae is a commonly used lower eukaryotichost microorganism. Others include Schizosaccharomyces pombe (Beach andNurse, Nature, 290:140 (1981); EP 139,383 published May 2, 1985);Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737 (1983)), K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van denBerg et al., Bio/Technology, 8:135 (1990)), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070;Sreekrishna et al., J. Basic Microbiol., 28:265-278 (1988)); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc.Natl. Acad. Sci. USA, 76:5259-5263 (1979)); Schwanniomyces such asSchwanniomyces occidentalis (EP 394,538 published Oct. 31, 1990); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357 published Jan. 10, 1991), and Aspergillus hosts such as A.nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289(1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al., Proc.Natl. Acad. Sci. USA, 81:1470-1474 (1984)) and A. niger (Kelly andHynes, EMBO J., 4:475479 (1985)). Methylotropic yeasts are suitableherein and include, but are not limited to, yeast capable of growth onmethanol selected from the genera consisting of Hansenula, Candida,Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list ofspecific species that are exemplary of this class of yeasts may be foundin C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated stretches ofconsecutive amino acids or polypeptides of interest are derived frommulticellular organisms. Examples of invertebrate cells include insectcells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.Examples of useful mammalian host cell lines include Chinese hamsterovary (CHO) and COS cells. More specific examples include monkey kidneyCV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonickidney line (293 or 293 cells subcloned for growth in suspensionculture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamsterovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

2. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the stretch ofconsecutive amino acids or polypeptides of interest may be inserted intoa replicable vector for cloning (amplification of the DNA) or forexpression. Various vectors are publicly available. The vector may, forexample, be in the form of a plasmid, cosmid, viral particle, or phage.The appropriate nucleic acid sequence may be inserted into the vector bya variety of procedures. In general, DNA is inserted into an appropriaterestriction endonuclease site(s) using techniques known in the art.Vector components generally include, but are not limited to, one or moreof a signal sequence, an origin of replication, one or more markergenes, an enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques which are known tothe skilled artisan.

The stretches of consecutive amino acids or polypeptides of interest maybe produced recombinantly not only directly, but also as a fusionpolypeptide with a heterologous polypeptide, which may be a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide. In general, the signalsequence may be a component of the vector, or it may be a part of theencoding DNA that is inserted into the vector. The signal sequence maybe a prokaryotic signal sequence selected, for example, from the groupof the alkaline phosphatase, penicillinase, lpp, or heat-stableenterotoxin II leaders. For yeast secretion the signal sequence may be,e.g., the yeast invertase leader, alpha factor leader (includingSaccharomyces and Kluyveromyces alpha-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2mu plasmid origin is suitable for yeast,and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) areuseful for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theencoding nucleic acid, such as DHFR or thymidine kinase. An appropriatehost cell when wild-type DHFR is employed is the CHO cell line deficientin DHFR activity, prepared and propagated as described by Urlaub et al.,Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection genefor use in yeast is the trp1 gene present in the yeast plasmid YRp7(Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141(1979); Tschemper et al., Gene, 10:157 (1980)). The trp1 gene provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones,Genetics, 85:12 (1977)).

Expression and cloning vectors usually contain a promoter operablylinked to the encoding nucleic acid sequence to direct mRNA synthesis.Promoters recognized by a variety of potential host cells are wellknown. Promoters suitable for use with prokaryotic hosts include thebeta-lactamase and lactose promoter systems (Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776), and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)).Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the encoding DNA.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Re.g., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

Transcription from vectors in mammalian host cells is controlled, forexample, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the stretches of consecutive amino acidsor polypeptides of interest by higher eukaryotes may be increased byinserting an enhancer sequence into the vector. Enhancers are cis-actingelements of DNA, usually about from 10 to 300 bp, that act on a promoterto increase its transcription. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, alpha-fetoprotein, andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. The enhancer may be spliced into thevector at a position 5′ or 3′ to the coding sequence, but is preferablylocated at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding stretches of consecutive amino acids orpolypeptides of interest.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of stretches of consecutive amino acids or polypeptides inrecombinant vertebrate cell culture are described in Gething et al.,Nature 293:620-625 (1981); Mantei et al., Nature, 281:4046 (1979); EP117,060; and EP 117,058.

3. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencestretches of consecutive amino acids or polypeptides of interest oragainst a synthetic peptide based on the DNA sequences provided hereinor against exogenous sequence fused to DNA encoding a stretch ofconsecutive amino acids or polypeptide of interest and encoding aspecific antibody epitope.

4. Purification of Polypeptide

Forms of the stretches of consecutive amino acids or polypeptides ofinterest may be recovered from culture medium or from host cell lysates.If membrane-bound, it can be released from the membrane using a suitabledetergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cellsemployed in expression of the stretches of consecutive amino acids orpolypeptides of interest can be disrupted by various physical orchemical means, such as freeze-thaw cycling, sonication, mechanicaldisruption, or cell lysing agents.

It may be desired to purify the stretches of consecutive amino acids orpolypeptides of interest from recombinant cell proteins or polypeptides.The following procedures are exemplary of suitable purificationprocedures: by fractionation on an ion-exchange column; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;protein A Sepharose columns to remove contaminants such as IgG; andmetal chelating columns to bind epitope-tagged forms. Various methods ofprotein purification may be employed and such methods are known in theart and described for example in Deutscher, Methods in Enzymology, 182(1990); Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York (1982). The purification step(s) selected willdepend, for example, on the nature of the production process used andthe particular stretches of consecutive amino acids or polypeptides ofinterest produced.

Example of Expression of Stretch of Consecutive Amino Acids orPolypeptide Component of Interest in E. coli

The DNA sequence encoding the desired amino acid sequence of interest orpolypeptide is initially amplified using selected PCR primers. Theprimers should contain restriction enzyme sites which correspond to therestriction enzyme sites on the selected expression vector. A variety ofexpression vectors may be employed. An example of a suitable vector ispBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977))which contains genes for ampicillin and tetracycline resistance. Thevector is digested with restriction enzyme and dephosphorylated. The PCRamplified sequences are then ligated into the vector. The vector willpreferably include sequences which encode for an antibiotic resistancegene, a trp promoter, a polyhis leader (including the first six STIIcodons, polyhis sequence, and enterokinase cleavage site), the specificamino acid sequence of interest/polypeptide coding region, lambdatranscriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized amino acid sequence of interest or polypeptide canthen be purified using a metal chelating column under conditions thatallow tight binding of the protein.

The primers can contain restriction enzyme sites which correspond to therestriction enzyme sites on the selected expression vector, and otheruseful sequences providing for efficient and reliable translationinitiation, rapid purification on a metal chelation column, andproteolytic removal with enterokinase. The PCR-amplified, poly-Histagged sequences can be ligated into an expression vector used totransform an E. coli host based on, for example, strain 52 (W3110fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq). Transformants can firstbe grown in LB containing 50 mg/ml carbenicillin at 30° C. with shakinguntil an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100fold into C RAP media (prepared by mixing 3.57 g (NH₄)₂SO₄, 0.71 gsodium citrate-2H₂O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 gSheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3,0.55% (w/v) glucose and 7 mM MgSO₄) and grown for approximately 20-30hours at 30° C. with shaking. Samples were removed to verify expressionby SDS-PAGE analysis, and the bulk culture is centrifuged to pellet thecells. Cell pellets were frozen until purification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) wasresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionwas stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution wascentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant was diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. Depending the clarified extract was loaded onto a 5mil Qiagen Ni-NTA metal chelate column equilibrated in the metal chelatecolumn buffer. The column was washed with additional buffer containing50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein waseluted with buffer containing 250 mM imidazole. Fractions containing thedesired protein were pooled and stored at 4.degree. C. Proteinconcentration was estimated by its absorbance at 280 nm using thecalculated extinction coefficient based on its amino acid sequence.

Expression of Stretch of Consecutive Amino Acids or Polypeptides inMammalian Cells

This general example illustrates a preparation of a glycosylated form ofa desired amino acid sequence of interest or polypeptide component byrecombinant expression in mammalian cells.

The vector pRK5 (see EP 307,247, published Mar. 15, 1989) can beemployed as the expression vector. Optionally, the encoding DNA isligated into pRK5 with selected restriction enzymes to allow insertionof the DNA using ligation methods such as described in Sambrook et al.,supra.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg of theligated vector DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell 31:543 (1982)] and dissolved in 500 μl of I mMTris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂ To this mixture is added, dropwise,500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and aprecipitate is allowed to form for 10 minutes at 25° C. The precipitateis suspended and added to the 293 cells and allowed to settle for aboutfour hours at 37° C. The culture medium is aspirated off and 2 ml of 20%glycerol in PBS is added for 30 seconds. The 293 cells are then washedwith serum free medium, fresh medium is added and the cells areincubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of amino acid sequence of interest or polypeptide component.The cultures containing transfected cells may undergo further incubation(in serum free medium) and the medium is tested in selected bioassays.

In an alternative technique, the nucleic acid amino acid sequence ofinterest or polypeptide component may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg of the ligated vector isadded. The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed amino acid sequence ofinterest or polypeptide component can then be concentrated and purifiedby any selected method, such as dialysis and/or column chromatography.

In another embodiment, the amino acid sequence of interest orpolypeptide component can be expressed in CHO cells. The amino acidsequence of interest or polypeptide component can be transfected intoCHO cells using known reagents such as CaPO₄ or DEAE-dextran. Asdescribed above, the cell cultures can be incubated, and the mediumreplaced with culture medium (alone) or medium containing a radiolabelsuch as ³⁵S-methionine. After determining the presence of amino acidsequence of interest or polypeptide component, the culture medium may bereplaced with serum free medium. Preferably, the cultures are incubatedfor about 6 days, and then the conditioned medium is harvested. Themedium containing the expressed amino acid sequence of interest orpolypeptide component can then be concentrated and purified by anyselected method.

Epitope-tagged amino acid sequence of interest or polypeptide componentmay also be expressed in host CHO cells. The amino acid sequence ofinterest or polypeptide component may be subcloned out of a pRK5 vector.The subclone insert can undergo PCR to fuse in frame with a selectedepitope tag such as a poly-his tag into a Baculovirus expression vector.The poly-his tagged amino acid sequence of interest or polypeptidecomponent insert can then be subcloned into a SV40 driven vectorcontaining a selection marker such as DHFR for selection of stableclones. Finally, the CHO cells can be transfected (as described above)with the SV40 driven vector. Labeling may be performed, as describedabove, to verify expression. The culture medium containing the expressedpoly-His tagged amino acid sequence of interest or polypeptide componentcan then be concentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

In an embodiment the amino acid sequence of interest or polypeptidecomponent are expressed as an IgG construct (immunoadhesin), in whichthe coding sequences for the soluble forms (e.g. extracellular domains)of the respective proteins are fused to an IgG1 constant region sequencecontaining the hinge, CH2 and CH2 domains and/or is a poly-His taggedform.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used in expression in CHOcells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Expression of Stretch of Consecutive Amino Acids or Polypeptides inYeast

The following method describes recombinant expression of a desired aminoacid sequence of interest or polypeptide component in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of a stretch of consecutive amino acids from theADH2/GAPDH promoter. DNA encoding a desired amino acid sequence ofinterest or polypeptide component, a selected signal peptide and thepromoter is inserted into suitable restriction enzyme sites in theselected plasmid to direct intracellular expression of the amino acidsequence of interest or polypeptide component. For secretion, DNAencoding the stretch of consecutive amino acids can be cloned into theselected plasmid, together with DNA encoding the ADH2/GAPDH promoter,the yeast alpha-factor secretory signal/leader sequence, and linkersequences (if needed) for expression of the stretch of consecutive aminoacids.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant amino acid sequence of interest or polypeptide component cansubsequently be isolated and purified by removing the yeast cells fromthe fermentation medium by centrifugation and then concentrating themedium using selected cartridge filters. The concentrate containing theamino acid sequence of interest or polypeptide component may further bepurified using selected column chromatography resins.

Expression of Stretches of Stretch of Consecutive Amino Acids orPolypeptides in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of stretches ofconsecutive amino acids in Baculovirus-infected insect cells.

The desired nucleic acid encoding the stretch of consecutive amino acidsis fused upstream of an epitope tag contained with a baculovirusexpression vector. Such epitope tags include poly-his tags andimmunoglobulin tags (like Fc regions of IgG). A variety of plasmids maybe employed, including plasmids derived from commercially availableplasmids such as pVL1393 (Novagen). Briefly, the amino acid sequence ofinterest or polypeptide component or the desired portion of the aminoacid sequence of interest or polypeptide component (such as the sequenceencoding the extracellular domain of a transmembrane protein) isamplified by PCR with primers complementary to the 5′ and 3′ regions.The 5′ primer may incorporate flanking (selected) restriction enzymesites. The product is then digested with those selected restrictionenzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression is performed as described byO'Reilley et al., Baculovirus expression vectors: A laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged amino acid sequence of interest or polypeptidecomponent can then be purified, for example, by Ni²⁺-chelate affinitychromatography as follows. Extracts are prepared from recombinantvirus-infected Sf9 cells as described by Rupert et al., Nature,362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended insonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA; 10%Glycerol; 0.1% NP40; 0.4 M KCl), and sonicated twice for 20 seconds onice. The sonicates are cleared by centrifugation, and the supernatant isdiluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%Glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% Glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged sequence are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) amino acidsequence can be performed using known chromatography techniques,including for instance, Protein A or Protein G column chromatography.

Fc containing constructs of proteins can be purified from conditionedmedia as follows. The conditioned media is pumped onto a 5 ml Protein Acolumn (Pharmacia) which is equilibrated in 20 mM Na phosphate buffer,pH 6.8. After loading, the column is washed extensively withequilibration buffer before elution with 100 mM citric acid, pH 3.5. Theeluted protein is immediately neutralized by collecting 1 ml fractionsinto tubes containing 275 mL of 1 M Tris buffer, pH 9. The highlypurified protein is subsequently desalted into storage buffer asdescribed above for the poly-His tagged proteins. The homogeneity of theproteins is verified by SDS polyacrylamide gel (PEG) electrophoresis andN-terminal amino acid sequencing by Edman degradation.

Examples of Pharmaceutical Compositions

Non-limiting examples of such compositions and dosages are set forth asfollows:

Compositions comprising a compound comprising a stretch of consecutiveamino acids which comprises consecutive amino acids having the sequenceof etanercept (e.g. Enbrel) may comprise mannitol, sucrose, andtromethamine. In an embodiment, the composition is in the form of alyophilizate. In an embodiment, the composition is reconstituted with,for example, Sterile Bacteriostatic Water for Injection (BWFI), USP(containing 0.9% benzyl alcohol). In an embodiment the compound isadministered to a subject for reducing signs and symptoms, inducingmajor clinical response, inhibiting the progression of structuraldamage, and improving physical function in subjects with moderately toseverely active rheumatoid arthritis. The compound may be initiated incombination with methotrexate (MTX) or used alone. In an embodiment thecompound is administered to a subject for reducing signs and symptoms ofmoderately to severely active polyarticular-course juvenile rheumatoidarthritis in subjects who have had an inadequate response to one or moreDMARDs. In an embodiment the compound is administered to a subject forreducing signs and symptoms, inhibiting the progression of structuraldamage of active arthritis, and improving physical function in subjectswith psoriatic arthritis. In an embodiment the compound is administeredto a subject for reducing signs and symptoms in subjects with activeankylosing spondylitis. In an embodiment the compound is administered toa subject for the treatment of chronic moderate to severe plaquepsoriasis. In an embodiment wherein the subject has rheumatoidarthritis, psoriatic arthritis, or ankylosing spondylitis the compoundis administered at 25-75 mg per week given as one or more subcutaneous(SC) injections. In a further embodiment the compound is administered at50 mg per week in a single SC injection. In an embodiment wherein thesubject has plaque psoriasis the compound is administered at 25-75 mgtwice weekly or 4 days apart for 3 months followed by a reduction to amaintenance dose of 25-75 mg per week. In a further embodiment thecompound is administered at a dose of at 50 mg twice weekly or 4 daysapart for 3 months followed by a reduction to a maintenance dose of 50mg per week. In an embodiment the dose is between 2× and 100× less thanthe doses set forth herein. In an embodiment wherein the subject hasactive polyarticular-course JRA the compound may be administered at adose of 0.2-1.2 mg/kg per week (up to a maximum of 75 mg per week). In afurther embodiment the compound is administered at a dose of 0.8 mg/kgper week (up to a maximum of 50 mg per week). In some embodiments thedose is between 2× and 100× less than the doses set forth hereinabove.

Compositions comprising a compound comprising a stretch of consecutiveamino acids which comprises consecutive amino acids having the sequenceof infliximab (e.g. Remicade) may comprise sucrose, polysorbate 80,monobasic sodium phosphate, monohydrate, and dibasic sodium phosphate,dihydrate. Preservatives are not present in one embodiment. In anembodiment, the composition is in the form of a lyophilizate. In anembodiment, the composition is reconstituted with, for example, Waterfor Injection (BWFI), USP. In an embodiment the pH of the composition is7.2 or is about 7.2. In one embodiment the compound is administered isadministered to a subject with rheumatoid arthritis in a dose of 2-4mg/kg given as an intravenous infusion followed with additional similardoses at 2 and 6 weeks after the first infusion then every 8 weeksthereafter. In a further embodiment the compound is administered in adose of 3 mg/kg given as an intravenous infusion followed withadditional similar doses at 2 and 6 weeks after the first infusion thenevery 8 weeks thereafter. In an embodiment the dose is adjusted up to 10mg/kg or treating as often as every 4 weeks. In an embodiment thecompound is administered in combination with methotrexate. In oneembodiment the compound is administered is administered to a subjectwith Crohn's disease or fistulizing Crohn's disease at dose of 2-7 mg/kggiven as an induction regimen at 0, 2 and 6 weeks followed by amaintenance regimen of 4-6 mg/kg every 8 weeks thereafter for thetreatment of moderately to severely active Crohn's disease orfistulizing disease. In a further embodiment the compound isadministered at a dose of 5 mg/kg given as an induction regimen at 0, 2and 6 weeks followed by a maintenance regimen of 5 mg/kg every 8 weeksthereafter for the treatment of moderately to severely active Crohn'sdisease or fistulizing disease. In an embodiment the dose is adjusted upto 10 mg/kg. In one embodiment the compound is administered to a subjectwith ankylosing spondylitis at a dose of 2-7 mg/kg given as anintravenous infusion followed with additional similar doses at 2 and 6weeks after the first infusion, then every 6 weeks thereafter. In afurther embodiment the compound is administered at a dose of 5 mg/kggiven as an intravenous infusion followed with additional similar dosesat 2 and 6 weeks after the first infusion, then every 6 weeksthereafter. In one embodiment the compound is administered to a subjectwith psoriatic arthritis at a dose of 2-7 mg/kg given as an intravenousinfusion followed with additional similar doses at 2 and 6 weeks afterthe first infusion then every 8 weeks thereafter. In a furtherembodiment the compound is administered at a dose of 5 mg/kg given as anintravenous infusion followed with additional similar doses at 2 and 6weeks after the first infusion then every 8 weeks thereafter. In anembodiment the compound is administered with methotrexate. In oneembodiment the compound is administered to a subject with ulcerativecolitis at a dose of 2-7 mg/kg given as an induction regimen at 0, 2 and6 weeks followed by a maintenance regimen of 2-7 mg/kg every 8 weeksthereafter for the treatment of moderately to severely active ulcerativecolitis. In a further embodiment the compound is administered to asubject with ulcerative colitis at a dose of 5 mg/kg given as aninduction regimen at 0, 2 and 6 weeks followed by a maintenance regimenof 5 mg/kg every 8 weeks thereafter. In some embodiments the dose isbetween 2× and 100× less than the doses set forth hereinabove fortreating the indivisual diseases.

In each of the embodiments of the compositions described herein, thecompositions, when in the form of a lyophilizate, may be reconstitutedwith, for example, sterile aqueous solutions, sterile water, SterileWater for Injections (USP), Sterile Bacteriostatic Water for Injections(USP), and equivalents thereof known to those skilled in the art.

It is understood that in administration of any of the instant compounds,the compound may be administered in isolation, in a carrier, as part ofa pharmaceutical composition, or in any appropriate vehicle.

Dosage

It is understood that where a dosage range is stated herein, e.g. 1-10mg/kg per week, the invention disclosed herein also contemplates eachinteger dose, and tenth thereof, between the upper and lower limits. Inthe case of the example given, therefore, the invention contemplates1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4 etc. mg/kg up to 10 mg/kg.

In embodiments, the compounds of the present invention can beadministered as a single dose or may be administered as multiple doses.

In general, the daily dosage for treating a disorder or conditionaccording to the methods described above will generally range from about0.01 to about 10.0 mg/kg body weight of the subject to be treated.

Variations based on the aforementioned dosage ranges may be made by aphysician of ordinary skill taking into account known considerationssuch as the weight, age, and condition of the person being treated, theseverity of the affliction, and the particular route of administrationchosen.

It is also expected that the compounds disclosed will effect cooperativebinding with attendant consequences on effective dosages required.

Pharmaceuticals

The term “pharmaceutically acceptable carrier” is understood to includeexcipients, carriers or diluents. The particular carrier, diluent orexcipient used will depend upon the means and purpose for which theactive ingredient is being applied.

For parenteral administration, solutions containing a compound of thisinvention or a pharmaceutically acceptable salt thereof in sterileaqueous solution may be employed. Such aqueous solutions should besuitably buffered if necessary and the liquid diluent first renderedisotonic with sufficient saline or glucose. These particular aqueoussolutions are especially suitable for intravenous, intramuscular,subcutaneous and intraperitoneal administration. The sterile aqueousmedia employed are all readily available by standard techniques known tothose skilled in the art.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions. The preferred form depends on the intended mode ofadministration and therapeutic application. Some compositions are in theform of injectable or infusible solutions. A mode of administration isparenteral (e.g., intravenous, subcutaneous, intraperitoneal,intramuscular). In an embodiment, the compound is administered byintravenous infusion or injection. In another embodiment, the compoundis administered by intramuscular or subcutaneous injection.

For therapeutic use, the compositions disclosed here can be administeredin various manners, including soluble form by bolus injection,continuous infusion, sustained release from implants, oral ingestion,local injection (e.g. intracrdiac, intramuscular), systemic injection,or other suitable techniques well known in the pharmaceutical arts.Other methods of pharmaceutical administration include, but are notlimited to oral, subcutaneously, transdermal, intravenous, intramuscularand parenteral methods of administration. Typically, a solublecomposition will comprise a purified compound in conjunction withphysiologically acceptable carriers, excipients or diluents. Suchcarriers will be nontoxic to recipients at the dosages andconcentrations employed. The preparation of such compositions can entailcombining a compound with buffers, antioxidants, carbohydrates includingglucose, sucrose or dextrins, chelating agents such as EDTA, glutathioneand other stabilizers and excipients. Neutral buffered saline or salinemixed with conspecific serum albumin are exemplary appropriate diluents.The product can be formulated as a lyophilizate using appropriateexcipient solutions (e.g., sucrose) as diluents.

Other derivatives comprise the compounds/compositions of this inventioncovalently bonded to a nonproteinaceous polymer. The bonding to thepolymer is generally conducted so as not to interfere with the preferredbiological activity of the compound, e.g. the binding activity of thecompound to a target. The nonproteinaceous polymer ordinarily is ahydrophilic synthetic polymer, i.e., a polymer not otherwise found innature. However, polymers which exist in nature and are produced byrecombinant or in vitro methods are useful, as are polymers which areisolated from nature. Hydrophilic polyvinyl polymers fall within thescope of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone.Particularly useful are polyalkylene ethers such as polyethylene glycol,polypropylene glycol, polyoxyethylene esters or methoxy polyethyleneglycol; polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, andblock copolymers of polyoxyethylene and polyoxypropylene (Pluronics);polymethacrylates; carbomers; branched or unbranched polysaccharideswhich comprise the saccharide monomers D-mannose, D- and L-galactose,fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,D-galacturontc acid, D-mannuronic acid (e.g. polymannuronic acid, oralginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminicacid including homopolysaccharides and heteropolysaccharides such aslactose, amylopectin, starch, hydroxyethyl starch, amylose, dextransulfate, dextran, dextrins, glycogen, or the polysaccharide subunit ofacid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugaralcohols such as polysorbitol and polymannitol; as well as heparin orheparon.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a compound of the invention. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the compound may vary according tofactors such as the disease state, age, sex, and weight of theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the compound are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

All combinations of the various elements disclosed herein are within thescope of the invention.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS Example 1: TNR1B-alkyne-azide-Fc6

TNR1B-alkyne-azide-Fc6 was prepared via the reaction of alkyne-modifiedTNR1B (TNF receptor 1B) with azide-modified Fc6 as follows.TNR1B-azide-alkyne-Fc6 is prepared using the same principles via thereaction of azide-modified TNR1B with alkyne-modified Fc6.

Alkyne-modified TNFR1B (TNR1B-Alk) was prepared by cleavage ofTNR1B-intein (TNR1B-Mth fusion protein) with cystyl-propargylamide,HSCH₂CH[NH₂]CONHCH₂C≡CH₃ (FIG. 1) and azide-modified TNR1B (TNR1B-Az)was prepared by cleavage of TNR1B-intein with cystyl-3-azidopropylamide,HSCH₂CH[NH₂]CONH (CH₂)₃NH₂.

TNR1B-intein and Fc6 are described in U.S. Ser. No. 11/982,085,published Oct. 16, 2008, the whole of which is incorporated herein byreference.

TNR1B-intein fusion protein was produced using vector pCDNA3-TNR1B-Mth,the sequence of which is shown in SEQ ID NO: 100. The pre-TNR1B-inteinchimeric polypeptide that is initially expressed, containing the TNR1Bextracellular domain joined at its C-terminus by a peptide bond to theN-terminus of an Mth RIR1 self-splicing intein at the autocleavage site,is shown in SEQ ID NO: 101. Cleavage of the homologous TNR signalsequences by the cellular signal peptidase provides the matureTNR1B-intein fusion protein that is secreted into the cell culturefluid, the sequence of which is shown in SEQ ID NO: 102.

Fc6 protein was expressed using vector pCDNA3-SHH-IgG1-Fc11, thesequence of which is shown in SEQ ID NO: 103. The pre-Fc6 polypeptidethat is initially expressed is shown in SEQ ID NO: 104. Cleavage of theheterologous sonic hedgehog (SHH) signal sequences by the cellularsignal peptidase provides the mature Fc6 protein that is secreted intothe cell culture fluid, the sequence of which is shown in SEQ ID NO:105.

Protein production was executed by transient expression in CHO-DG44cells, adapted to serum-free suspension culture. Transient transfectionswere done with polyethylenimine as transfection agent, complexed withDNA, under high density conditions as described by Rajendra et al., J.Biotechnol. 153, 22-26 (2011). Seed train cultures were maintained inTubeSpin® bioreactor 50 tubes obtained from TPP (Trasadingen, CH) andscaled up in volume to generate sufficient biomass for transfection.Transfections were carried out in cultures of 0.5-1.0 L. Cultures atthis scale were maintained in 2 L or 5 L Schott-bottles with aventilated cap. The bottles were shaken at 180 rpm in a Kühner incubatorshaker with humidification and CO₂ control at 5%. The cell culture fluidwas harvested after 10 days, centrifuged and sterile-filtered, prior topurification.

Cystyl-propargylamide and cystyl-3-azidopropylamide were prepared asfollows. Boc-Cys(Trt)-OH, (C₆H₅)₃CSCH₂CH[NHCO₂C(CH₃)₃]CO₂H;propargylamine, HC≡CCH₂NH; 3-azidopropylamine, NH₂CH₂CH₂CH₂N₃; EDC,N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; and HOBt,1-Hydroxybenzotriazole, and were obtained from AnaSpec (Freemont,Calif.) or CPC Scientific (San Jose, Calif.). All other chemicals wereobtained from Sigma-Aldrich (St. Louis, Mo.). For the synthesis ofcystyl-propargylamide, a solution of Boc-Cys(Trt)-OH (100 mM) andpropargylamine (100 mM) in CH2Cl2 was made 100 mM each in EDC, HOBt, andtriethylamine. For the synthesis of cystyl-3-azidopropylamide,3-azidopropylamine (100 mM) was substituted for propargylamine. Bothreactions were worked up by the following procedure. After stirringovernight at room temperature, the reaction was stopped with an excessof saturated NaHCO3 in water, extracted with CH2Cl2, dried over MgSO4,filtered, evaporated, and purified by column chromatography. To removethe Boc/Trt protecting groups, the intermediate product was dissolved ata concentration of 0.05M in TFA/triisopropylsilane/H2O (90:5:5) andstirred for 30 minutes at room temperature. The reaction was then driedby evaporation and extracted with CH2Cl2. The organic layer was thenextracted with water, yielding the final cystyl-propargylamide productas a yellowish oil, and the final cystyl-3-azidopropylamide product as ayellowish solid.

To prepare the alkyne-modified TNR1B (FIG. 1) or the azide-modifiedTNR1B, the TNR1B-intein protein in the cell culture fluid was applied toa column packed with chitin beads obtained from New England BioLabs(Beverley, Mass.) that was pre-equilibrated with buffer A (20 mMTris-HCl, 500 mM NaCl, pH 7.5). Unbound protein was washed from thecolumn with buffer A. Cleavage was initiated by rapidly equilibratingthe chitin resin in buffer B (20 mM Tris-HCl, 500 mM NaCl, pH 8.0)containing either 50 mM cystyl-propargylamide (for alkyne-modifiedTNR1B) or 50 mM cystyl-3-azidopropylamide (for azide-modified TNR1B) andincubation was carried out for 24 to 96 hours at room temperature. Thecleaved alkyne-modified TNR1B (SEQ ID NO: 106) or azide-modified TNR1Bproteins (SEQ ID NO: 107) were eluted from the column with buffer A,concentrated using an Amicon Ultracel-3 Centrifugal Filter Unit fromMillipore (Billerica, Mass.), dialyzed against Dulbecco's phosphatebuffered saline without Ca or Mg salts (PBS) obtained from the UCSF CellCulture Facility (San Francisco, Calif.), and stored at 4° C. prior touse.

FIG. 2 shows SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysisof the alkyne-modified TNR1B, compared with cysteine-modified TNR1B (SEQID NO: 108) prepared using 50 mM cysteine instead ofcystyl-propargylamide. SDS-PAGE was carried out using NuPAGE® NovexBis-Tris Midi Gels (10%) obtained from Invitrogen (Carlsbad, Calif.).Proteins were visualized using Silver Stain Plus or Bio-Safe CoomassieStain obtained from Bio-Rad (Hercules, Calif.). The alkyne-modifiedTNR1B (lane 3) and the cysteine-modified TNR1B (lane 1) had the sameMr˜43,000. In addition, the alkyne-modified TNR1B had comparablebiological activity to cysteine-modified TNR1B as measured using a HumansTNFRII/TNFRSF1B Quantikine ELISA obtained from R&D Systems(Minneapolis, Minn.). Preparations of the cysteine-modified TNR1B (lane2), alkyne-modified TNR1B (lane 4), or thioester-modified TNR1B (SEQ IDNO: 109) (lane 5) made in the presence of 50 mM MESNA had a similar Mr,but had less than 5% of the biological activity observed forpreparations made in the absence of MESNA. Thus, alkyne-modified TNR1Bprepared in the absence of MESNA was employed in further studies.

Azide-modified Fc6 (Az-Fc6) was prepared by the reaction of Fc6 proteinwith various azide-containing peptide thioesters (FIG. 3) andazide-containing PEG thioesters (FIG. 4). Alkyne-modified Fc6 (Alk-Fc6)was prepared by the reaction of alkyne-containing thioesters with Fc6protein.

Recombinant Fc6 protein was expressed in Chinese hamster ovary (CHO)cells as described for TNR1B-intein (see above) and purified by ProteinA affinity chromatography. The culture supernatant was applied to acolumn packed with rProtein A Fast Flow from Pharmacia (Uppsala, Sweden)pre-equilibrated with PBS. The column was washed extensively with PBSand the Fc6 protein then eluted with 0.1 M glycine buffer pH 2.7.Fractions were collected into tubes containing 0.05 vol/vol of 1.0 MTris-HCl pH 9.0 (giving a final pH of 7.5), pooled, dialyzed againstPBS, and stored at 4° C. prior to use.

Table 1 shows representative azide-containing and alkyne-containingpeptide/PEG thioesters. Thioesters were synthesized by an Fmoc/t-Butylsolid-phase strategy on a 2-chlorotrityl chloride resin preloaded withthe Fmoc-Thr(tBu)-OH. Amino acid derivatives were obtained from CPCScientific (Sunnyvale, Calif.), Fmoc-PEG_(n)-OH derivatives wereobtained from Quanta BioDesign (Powell, Ohio), and2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate(HBTU), dichloromethane (DCM), trichloroacetic acid (TFA),N,N′-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt),N,N′-diisopropylethylamine (DIEA) and triisopropylsilane (TIS) wereobtained from Sigma (St. Louis, Mo.). The standard HBTU activation wasemployed for peptide elongation. Peptides containing PEG required theinsertion of a Fmoc-PEG_(n)-OH. As a final step in peptide elongation,the terminal a-Fmoc (9-fluorenylmethoxycarbonyl) protecting group wasconverted to Boc (tert-butoxycarbonyl). The peptide resin was washedwith DCM and cleaved with 1% TFA/DCM to yield the fully protectedpeptide with a free carboxylic acid on the C-terminus. The thioester ofthe peptides was formed by treating the crude protected peptide withDIC/HOBt/DIEA and benzyl mercaptan or thiophenol in DCM overnight. Afterconcentration, the crude protected peptide thioester was precipitated bymultiple triturations with cold ether followed by centrifugation.Deprotection was carried out by treatment of the crude protected productwith 95:2.5:2.5 TFA/TIS/H₂O for 2 hours at room temperature. Afterprecipitation with ice-cold ether the deprotected peptide thioester waspurified by preparative RP-HPLC in a H₂O-acetonitrile (0.1% TFA) systemto afford the final product with 91-95% purity and the desired MS.

Azide-modified Fc6 and alkyne-modified Fc6 were prepared by nativechemical ligation as follows. 2-(N-morpholino)ethanesulfonic acid (MES)was obtained from Acros (Morris Plains, N.J.),tris(2-carboxyethyl)phosphine (TCEP) was obtained from Pierce (Rockford,Ill.), and 4-mercaptophenylacetic acid (MPAA) was obtained fromSigma-Aldrich (St. Louis, Mo.). Reactions were carried out by ligatingthe various thioesters shown in Table 1 with the Fc6 protein as follows.Reactions (100 uL) contained 50 mM MES buffer, pH 6.5, 0.8 mM TCEP, 10mM MPAA, 4 mg/ml of the peptide thioester, and 0.5 mg/ml of the Fc6protein. Following overnight incubation at room temperature, reactionswere adjusted to pH 7.0 with 0.05 vol/vol of 1.0 M Tris-HCl pH 9.0,purified using Protein A Magnetic Beads from New England BioLabs,dialyzed in 0.1 M phosphate pH 8.0, and concentrated.

FIG. 5 shows SDS-PAGE analysis demonstrating that Fc6 protein (lane 1)reacted quantitatively with azide-DKTHT-thioester to yield theAz-DKTHT-Fc6 protein (lane 2) and azide-PEG₄-DKTHT-thioester to yieldthe Az-PEG₄-DKTHT-Fc6 protein (lane 3). The sequence of the Az-DKTHT-Fc6protein is shown in SEQ ID NO: 110 and the sequence of theAz-PEG₄-DKTHT-Fc6 is shown in SEQ ID NO: 111. The PEG₄ oligomer gave anincremental size increase comparable to the 5 amino acid DKTHT

sequence. This shows that a single oxyethylene monomer unit makes acontribution to contour length similar to a single amino acid residue,consistent with their having comparable fully extended conformations of˜3.5 to 4 Å (Flory (1969) Statistical Mechanics of Chain Molecules(Interscience Publishers, New York).

TNR1B-alkyne-azide-Fc6 was prepared via the reaction of thealkyne-modified TNR1B with the Az-DKTHT-Fc6 protein (FIG. 6) and theAz-PEG₄-DKTHT-Fc6 protein (FIG. 7). Sodium phosphate, dibasic(anhydrous) and sodium phosphate, monobasic (monohydrate) were obtainedfrom Acros, TCEP was from Pierce, CuSO₄ (pentahydrate) was fromSigma-Aldrich, and Tris[1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine(TBTA) from AnaSpec (Freemont, Calif.). Reactions (60 uL) contained 0.1M sodium phoshate, pH 8.0, 1.0 mM CuSO₄, 2.0 mM TBTA, thealkyne-modified TNR1B (30 ug), and either the unmodified Fc6 protein,the Az-DKTHT-Fc6 protein, or the Az-PEG₄-DKTHT-Fc6 protein (10 ug).Reactions were initiated by the addition of 2.0 mM TCEP, and incubatedovernight at room temperature. The reaction products were purified usingProtein A Magnetic Beads to remove any unreacted alkyne-modified TNR1B.

FIG. 8 shows SDS-PAGE analysis of the TNR1B-alkyne-azide-Fc6 productsunder reducing conditions. In the absence of CuSO₄, TBTA and TCEP, bothAz-DKTHT-Fc6 (lane 2) and Az-PEG₄-DKTHT-Fc6 (lane 5) gave a single bandof Mr˜28-30,000 daltons (arrow d) corresponding to the inputazide-modified Fc6 proteins, with no sign of any product formation. Inaddition, there was no evidence of any carryover of the inputalkyne-modified TNR1B (shown in lane 1) following the Protein Apurification. However, in the presence of CuSO₄, TBTA and TCEP, thereaction between alkyne-modified TNR1B and Az-DKTHT-Fc6 (lane 3) and thereaction between alkyne-modified TNR1B and Az-PEG₄-DKTHT-Fc6 (lane 6)both yielded two new products of Mr˜75,000 daltons (arrow a) and ˜65,000daltons (arrow b). Reactions carried out using a preparation ofalkyne-modified TNR1B following buffer-exchange in 0.1 M phosphate pH8.0 to remove salt gave essentially similar reaction products with bothAz-DKTHT-Fc6 (lane 4) and Az-PEG₄-DKTHT-Fc6 (lane 6), although there wasa significant increase in the yield of the Mr˜75,000 dalton product overthe Mr˜65,000 dalton product.

FIG. 9 shows SDS-PAGE analysis comparing the TNR1B-alkyne-azide-Fc6reaction products (left panel) and the TNR1B-alkyne-azide-PEG₄-Fc6reaction products (right panel) with TNR1B-Fc fusion protein(etanercept). The TNR1B-alkyne-azide-Fc6 product of Mr˜75,000 daltons(lane 2), having the predicted sequence shown in SEQ ID NO: 112 joinedby the alkyne-azide non-peptidyl linker to SEQ ID NO: 113, and theTNR1B-alkyne-azide-PEG₄-Fc6 product of Mr˜75,000 daltons (lane 4),having the predicted sequence of shown in SEQ ID NO: 112 joined by thealkyne-azide non-peptidyl and PEG₄ linker to SEQ ID NO: 113, areessentially indistinguishable in size from etanercept (lanes 1, 3), thesequence of which is shown in SEQ ID NO: 114.

FIG. 10 shows SDS-PAGE analysis providing further evidence confirmingthe requirement of the alkyne and azide groups for reactivity. Reactionmixtures that contained alkyne-modified TNR1B with unmodified Fc6protein gave no reaction product (lane 2) compared with Fc6 alone (lane1), while alkyne-modified TNR1B with Az-DKTHT-Fc6 gave the expectedproducts (lane 4) compared with Az-DKTHT-Fc6 alone (lane 3). Again, nocarryover of the input alkyne-modified TNR1B (shown in lane 5) wasapparent following the Protein A purification.

The TNR1B-alkyne-azide-Fc6 products of FIG. 10 were furthercharacterized by sequencing of their tryptic peptide by LC-MS. FollowingSDS-PAGE, the gel was Coomassie stained and four gel regions wereexcised, corresponding to the Mr˜75,000 product (arrow a), the Mr˜65,000product (arrow b), the unstained region where alkyne-modified TNR1Bwould migrate (arrow c), and the unreacted Az-DKTHT-Fc6 protein ofMr˜28,000 (arrow d). The four gel slices were diced into small smallpieces (˜0.5-1.0 mm³) and processed as follows. Ammonium bicarbonate,acetonitrile, dithiothreitol, and iodoacetamide were obtained fromSigma-Aldrich, formic acid was obtained from Pierce, and porcine trypsin(sequencing grade) was obtained from Promega (Madison, Wis.). To removethe Coomassie stain, each gel slice was extracted with 200 uL of 25 mMNH₄HCO₃ in 50% acetonitrile by vortexing, centrifuged to remove thesupernatant, and dehydrated by adding acetonitrile for a few minutesuntil the gel pieces shrank and turned white. The acetonitrile wasdiscarded, and the gel slices dried in a Speed Vac (Savant Instruments,Farmingdale, N.Y.). Reduction and alkylation was then carried out byrehydrating the gel slices in 40 ul of 10 mM dithiothreitol in 25 mMNH₄HCO₃, vortexing, and incubated at 56° C. for 45 minutes. Thesupernatant was then discarded, 40 uL of 55 mM iodoacetamide in 25 mMNH₄HCO₃ was added, the gel slices vortexed and incubated in the dark for30 minutes at room temperature. The supernatant was discarded, the gelslices again dehydrated in acetonitrile and dried in a Speed Vac.Trypsin digestion was then carried out by rehydrating the gel slices in25 uL of trypsin (12.5 ug/mL) in 25 mM NH₄HCO₃ on ice for 60 minutes.Excess trypsin solution was then removed, the gel slices covered with 25mM NH₄HCO₃ and incubated at 37° C. overnight. The supernatant wasremoved, and the gel then extracted twice with 30 uL of 50%acetonitrile/0.1% formic acid in water. The organic extracts werecombined with the aqueous supernatant, reduced to a volume of 10 uL in aSpeed Vac, then analysed by LC-MS using a Q-Star Elite mass spectrometer(AB SCIEX, Foster City, Calif.).

FIG. 11 summarizes the characterization of the structure of theTNR1B-alkyne-azide-Fc6 reaction product by mass spectrometry. TheMr˜75,000 product, as expected for the full-lengthTNR1B-alkyne-azide-Fc6 product, contained peptides from both thealkyne-modified TNR1B and azide-modified Fc6 parent proteins. Inaddition, the peptide coverage of the alkyne-modified TNR1B sequence(upper panel) extended from the N-terminal region (EYYDQTAQMCCSK, aminoacids 22-34 of SEQ ID NO: 114) to the C-terminal region(SMAPGAVHLPQPVST, amino acids 186-200 of SEQ ID NO: 114). Similarly, thepeptide coverage of the azide-modified Fc6 protein sequence (lowerpanel) extended from the N-terminal region (DTLMISR, amino acids 76-83of SEQ ID NO: 113) to the C-terminal region (TTPPVLDSDGSFFLYSK, aminoacids 221-236 of SEQ ID NO: 113). In contrast, the Mr˜65,000 lacked theEYYDQTAQMCCSK (amino acids 22-34 of SEQ ID NO: 114) peptide, suggestingit was an N-terminally deleted version of the expected full-lengthTNR1B-alkyne-azide-Fc6 product. Sequences derived from the TNR1B proteinwere not detected in the unstained region of Mr˜43,000 where thealkyne-modified TNR1B would normally migrate (arrow c), while onlysequences derived from the Fc6 protein were detected in the unreactedAz-DKTHT-Fc6 protein of Mr˜28,000 (arrow d).

The TNR1B-alkyne-azide-Fc6 and TNR1B-alkyne-azide-PEG₄-Fc6 products ofFIG. 10 were further characterized for their biological activity bymeasuring their ability to bind TNF-α using surface plasmon resonance(SPR). Recombinant human TNF-α protein (carrier-free) was obtained fromR&D Systems and reconstituted in PBS. SPR studies were carried out usinga Biacore T100 instrument from Biacore AB (Uppsala, Sweden). Thesurface-bound ligands, TNR1B-alkyne-azide-Fc6 andTNR1B-alkyne-azide-PEG₄-Fc6, were immobilized onto a CM5 sensor chip,Series S, using a Amine Coupling Kit (BR-1000-50) obtained from GEHealthcare (Piscataway, N.J.) according to the manufacturer'sinstructions. Binding of TNF-α was carried out at 25° C. in 10 mM Hepesbuffer pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween-20. Binding wasevaluated in duplicate at TNF-α concentrations of 0.156 nM, 0.312 nM,0.625 nM, 1.25 nM, 2.5 nM, 5.0 nM, 10.0 nM, 20.0 nM and 40 nM. Data wasevaluated using Biacore T100 Evaluation Software, version 2.0.3.

FIG. 12 shows the kinetic binding curves for TNR1B-alkyne-azide-Fc6(left panel) and TNR1B-alkyne-azide-PEG₄-Fc6 (right panel). Bothproducts showed saturable TNF-α binding, and an excellent fit wasobtained employing a two-exponential model (Chi²˜0.05). Table 2summarizes the kinetic binding data. Approximately 40% of the bindingsites for each product were higher affinity, with a 1.6-fold greaterdissociation constant for TNR1B-alkyne-azide-PEG₄-Fc6 (K_(D)=1.86×10⁻¹⁰M) than for TNR1B-alkyne-azide-Fc6 (K_(D)=2.99×10⁻¹⁰ M). The remaining60% of the binding sites were of lower affinity, with the dissociationconstants about the same for TNR1B-alkyne-azide-PEG₄-Fc6(K_(D)=5.12×10⁻⁹ M) and TNR1B-alkyne-azide-Fc6 (K_(D)=5.17×10⁻⁹ M). Theassociation of the PEG₄ linker with increased high affinity binding, butequal low affinity binding, provides compelling evidence for a higherdegree of cooperative (two-handed) binding of TNF-α byTNR1B-alkyne-azide-PEG₄-Fc6 compared with TNR1B-alkyne-azide-Fc6.

TABLE 1 Azide-containing and Alkyne-Containing Thioesters Name FormulaMr MH⁻ Sequence Az- C₃₃H₄₇O₁₀N₁₁S 789.86 780.60 Azide-DKTHT- DKTHTthioester Az-PEG₄- C₄₄H₆₈O₁₅N₁₂S 1037.14 1038.20 Azide-PEG₄- DKTHTDKTHT- thioester Az-PEG₁₂- C₅₉H₉₈O₂₃N₁₂S 1375.55 1376.26 Azide-PEG₁₂-DKTHT DKTHT- thioester Az-PEG₂₄- C₈₃H₁₄₆O₃₅N₁₂S 1904.18 1904.80Azide-PEG₂₄- DKTHT DKTHT- thioester Az-PEG₃₆- C₁₀₇H₁₉₄O₄₇N₁₂S 2432.822434.40 Azide-PEG₃₆- DKTHT DKTHT- thioester Alk-PEG₁₂ C₅₃H₇₄O₁₅N₂S1011.22 1011.80 DIBAC-PEG₁₂- thioester Mr, relative molecular mass; MH⁻,monoisotypic mass value.

TABLE 2 TNF-α binding measured by surface plasmon resonanceSurface-bound ligand ka1 (1/Ms) kd1 (1/s) KD1 (M) Rmax1 ka2 (1/Ms) kd2(1/s) KD2 (M) Rmax2 Chi² TNR1B-Alk-Az-DKTHT-Fc6 1.252E−7 0.0037442.990E−10 2.5 5.176E−6 0.03392 6.553E−9 3.9 0.0514THR1B-Alk-Az-PEG4-DKTHT-Fc6 1.400E−7 0.002613 1.866E−10 3.0 5.129E−60.03021 5.890E−9 4.8 0.0503 Abbreviations: ka, on-rate (measured); kd,off-rate (measured); KD, dissociation constant (calculated).

Example 2: Fab′-Alkyne-Azide-Fc6

Fab′-alkyne-azide-Fc6 was prepared via the reaction ofcycloalkyne-modified Fab′ with azide-modified Fc6 as follows.

Adalimumab (Humira) was obtained as a liquid formulation (50 mg/ml) fromAbbott (Abbott Park, Ill.). The Fab′ fragment was prepared using IdesSprotease to first generate Fab′2 fragment followed by selectivereduction of the interchain disulfides with 2-mercaptoethylamine (FIG.13). Antibody (10 mg) was exchanged into cleavage buffer (50 mM sodiumphosphate, 150 mM NaCl, pH 6.6) using a Slide-A-Lyzer Mini DialysisUnit, 10K MWCO from Pierce (Rockford, Ill.), then incubated withhis-tagged recombinant IdeS immobilized on agarose beads (FragITMidiSpin column) from Genovis (Lund, Sweden) for 1 hour at roomtemperature with constant mixing. The beads were removed from the digestsolution by centrifugation, washed twice with cleavage buffer, and thedigest and wash solutions then combined and applied to a HiTrap ProteinA HP column from GE Life Sciences (Piscataway, N.J.) to remove Fc′fragment and undigested antibody. Flow-through fractions containing theFab′2 fragment were reduced to the Fab′ fragment by adding 1 mL aliquotsto a vial containing 6 mg 2-mercaptoethylamine (MEA) from Pierce.Reductions were carried out with 10 mM EDTA to minimize re-oxidation ofthe interchain disulfides. Following incubation at 37° C. for 110 min,excess MEA was removed by buffer-exchange into PBS containing 10 mM EDTAusing a PD-10 desalting column from GE Life Sciences (Piscataway, N.J.).The eluate containing the Fab′ fragment was concentrated using an AmiconUltracel-3 Centrifugal Filter Unit from Millipore (Billerica, Mass.).

FIG. 14 shows SDS-PAGE analysis of adalimumab after cleavage with IdeS(panel A), followed by Protein A chromatography and mild reduction withMEA (panel B). In the presence of a strong reducing agent(dithiothreitol) in the polyacrylamide gel, the whole antibody (lane 1)migrated as a heavy chain of Mr˜55,000 (arrow a) and a light chain ofMr˜25,000 (arrow d). IdeS cleaved the heavy chain (lane 2) into aC-terminal fragment of Mr˜29,000 (arrow b) and an N-terminal fragment ofMr˜26,000 (arrow c). The light chain and the N-terminal heavy chainfragment comprise the Fab′2 domain, while the C-terminal heavy chainfragment comprises the Fc′ domain. The Protein A column efficientlyremoved the Fc′ domain from the Fab′ domain (compare lane 2 with lanes 5and 6). Under non-reducing conditions, the Fab′2 domain migrated as asingle species of Mr˜110,000 (lane 3), while the Fab′ domain produced bymild reduction with MEA migrated as a single species of Mr˜55,000 (lane4). Under reducing conditions, the Fab′2 domain (lane 5) and the Fab′domain (lane 6) both yielded the same light chain (arrow d) andN-terminal heavy chain fragment (arrow c), as expected. Thus, the Fab′domain obtained by this procedure was essentially free of the Fab′2 andFc′ domains.

Cycloalkyne-modified Fab′ was prepared from the adalimumab Fab′ domainusing a bifunctional linker, DIBAC-PEG₁₂-Lys(Mal), which contains amaleimide group capable of reacting with the free thiol groups on theFab′ fragment (FIG. 15). DIBAC-PEG₁₂-Lys(Mal) was prepared using an Fmocsolid-phase synthesis strategy. Lys(Mtt)-Wang resin and succinimido3-maleimidopropanoate (Mpa-OSu) were obtained from CPC Scientific(Sunnyvale, Calif.), Fmoc-N-amido-dPEG₁₂-acid was obtained from QuantaBioDesign (Powell, Ohio), and5-(11,12-Didehydrodibenzo[b,f]azocin-5(6H)-yl)-5-oxopentanoic acid, anacid-functionalised aza-dibenzocyclooctyne (DIBAC-acid), was synthesizedas described by Debets, M. F. et al., Chem. Commun. 46, 97-99 (2010).Fmoc-N-amido-dPEG₁₂-acid and DIBAC-acid were sequentially reacted withLys(Mtt)-Wang resin to obtain DIBAC-PEG₁₂-Lys(Mtt)-Wang resin, thentreated with TFA/DCM/TIS(1:96:3) to remove the Mtt group. Thedeprotected resin was reacted with Mpa-OSu on the free amino group onthe lysine side chain to obtain DIBAC-PEG12-Lys(Mpa)-Wang resin.Following cleavage with TFA/water (95:5), the crude product was purifiedby preparative RP-HPLC to afford the DIBAC-PEG₁₂-Lys(Mal) product (DPKM)with 93% purity and the desired MS spectra.

FIG. 16 shows the chemical modification of adalimumab Fab′ fragment withthe DIBAC-PEG₁₂-Lys(Mal) linker and the purification of the resultingcycloalkyne-modified Fab′. For purification, reactions (0.535 mL) werecarried out in 0.1 M sodium phosphate at pH 7.0 and pH 7.4, eachcontaining 5 mg of Fab′ fragment and 10 mg of DIBAC-PEG₁₂-Lys(Mal).After 30 hours incubation at room temperature, the two reactions werecombined and buffered-exchanged into 20 mM sodium acetate, 20 mM NaCl,pH 5.5 using a PD-10 column. The eluate (3.5 mL) was applied to a HiTrapSP HP cation-exchange column from GE Life Sciences which retained allthe unmodified Fab′ and residual Fab′2. The flow-through fractions (5.5mL) containing the purified cycloalkyne-modified Fab′ (FIG. 16b ) werepooled, adjusted to pH 7.0 with 10×PBS (0.55 mL), and concentrated byaffinity chromatography on a Protein L column (Capto L) from GE LifeSciences. The cycloalkyne-modified Fab′ was eluted from the Protein Lcolumn with 0.1 M glycine HCl pH 2.7 (2.4 mL), neutralized with 1/20volume 1.0 M Tris HCl pH 9.0, buffered-exchanged into PBS using a PD-10column (3.5 mL) and concentrated using Amicon Ultracel-3 CentrifugalFilter Unit to a final volume of 70 uL at a concentration of 9.5 mg/mL.

Various azide-modified Fc6 proteins with PEG linkers of differentlengths were used in the preparation of the adalimumabFab′-cycloalkyne-azide-Fc6. Az-DKTHT-Fc6 (FIG. 3) andAz-DKTHT-PEG_(x)-Fc6 derivatives with x=12, 24, and 36 (FIG. 4) wereprepared in reactions (2 ml) that contained 50 mM MES pH 6.5, 0.8 mMTCEP, 10 mM MPAA, 5 mg/ml of each of the fourAz-DKTHT-PEG_(x)-thioesters, and 2.36 mg/ml of Fc6 protein. After 20hours at room temperature, the reactions were neutralized with 100 uL ofTris HCl pH 9.0, clarified by centrigugation at 12,000×g, and applied toa 1 ml HiTrap Protein A HP column. The columns were washed with 12 volof PBS, the azide-modified Fc6 proteins were then eluted with 0.1 Mglycine HCl pH 2.7 (2.0 mL), neutralized with 1/20 volume 1.0 M Tris HClpH 9.0, dialysed against three changes of PBS for 12 hours each using aSlide-A-Lyzer Mini Dialysis Unit, 10K MWCO, and concentrated usingAmicon Ultracel-3 Centrifugal Filter Units.

FIG. 17 shows analysis by SDS-PAGE under reducing conditions of the Fc6(lane 1) Az-DKTHT-Fc6 (lane 2), Az-DKTHT-PEG₁₂-Fc6 (lane 3),Az-DKTHT-PEG₂₄-Fc6 (lane 4), and Az-DKTHT-PEG₃₆-Fc6 (lane 5) proteins bySDS-PAGE. The Fc6 protein reacted quantitatively (>90%) with all fourthioesters, yielding a ladder of products of increasing size.

FIG. 18 shows analysis by size-exclusion chromatography (SEC) to confirmthat the four azide-modified Fc6 protein products had the same dimericstructure as the parent Fc6 molecule. SEC was carried out using aProminence HPLC System (Shimadzu Corp, Kyoto, Japan). TSKgel SuperSW3000 columns (4.6 mm×30 cm column, 4.6 mm×5 cm guard column) wereobtained from TOSOH Bioscience (Tokyo, Japan). Mobile phase, flow rate,column temperature, and detection wavelength used were 50 mM sodiumphosphate, 300 mM NaCl, pH 7.4, 0.35 mL/min., 30° C., and 280 nm,respectively. The four azide-modified Fc6 protein products displayed aretention time that decreased as the size of PEG linker increased,confirming their dimer structure. All four products also gaveessentially a single peak, demonstrating a two-handed structure in whichboth N-termini of the parent Fc6 dimer were modified by the PEG linkerthat was confirmed by SDS-PAGE analysis under non-reducing conditions(see below).

The cyclooctyne-modified Fab′ was reacted with all four azide-modifiedFc6 molecules (FIG. 19), yielding a family ofFab′-PEG_(y)-cycloalkyne-azide-PEG_(x)-Fc6 structures with arms ofincreasing length (FIG. 20). The overall lengths of the resulting armswere Fab′-PEG₁₂-Fc6 (for x=0, y=12), Fab′-PEG₂₄-Fc6 (for x=12, y=12),Fab′-PEG₃₆-Fc6 (for x=24, y=12), and Fab′-PEG₄₈-Fc6 (for x=36, y=12).The reactions (8 uL) were carried out in 0.1 M sodium phosphate pH 7.0overnight at room temperature with each of the four azide-modified Fc6proteins (2.5 mg/ml) in the presence or the absence of thecycloalkyne-modified Fab′ (5 mg/ml).

FIG. 21 shows SDS-PAGE analysis of the Fab′-cycloalkyne-azide-Fc6reaction under reducing and non-reducing conditions. In the absence ofthe cycloalkyne-modified Fab′ (lanes 5, 7, 9, and 11), all four of theazide-modified Fc6 proteins gave a single band on both reducing andnon-reducing gels, confirming their dimeric, two-handed handedstructure. In the presence of the cycloalkyne-modified Fab′ (lanes 4, 6,8, and 10), all four of the azide-modified Fc6 proteins were largelyconsumed in the resulting reaction. Under reducing conditions, all fourreactions gave a product with Mr˜57,000 to 62,000 (arrow a). The size ofthe Fab′-PEG₁₂-Fc6 product (lane 4) was approximately 1-2 kD greaterthan the wild-type adalimumab heavy chain (lane 1), while the sizes ofthe Fab′-PEG₂₄-Fc6 (lane 6), Fab′-PEG₃₆-Fc6 (lane 8), and Fab′-PEG₄₈-Fc6(lane 10) products further increased with the overall length of the PEGlinker. Under non-reducing conditions, two products were observed, afirst product of Mr˜155,000 to 160,000 (arrow a), and a second ofMr˜110,000 to 115,000 (arrow b). The larger Fab′-PEG₁₂-Fc6 product (lane4) was approximately 5 kD greater than the adalimumab whole antibody(lane 1), consistent with the expected two-handed product, while thelarger Fab′-PEG₂₄-Fc6 (lane 6), Fab′-PEG₃₆-Fc6 (lane 8), andFab′-PEG₄₈-Fc6 (lane 10) products still further increased in size as theoverall length of the PEG linker increased.

FIG. 22 shows analysis by SEC to confirm the two-handed structure (ie,two Fab′ hands attached to one Fc6 domain) of the larger reactionproduct with Mr˜155,000 to 160,000 of the Fab′-PEG₁₂-Fc6,Fab′-PEG₂₄-Fc6, Fab′-PEG₃₆-Fc6, and Fab′-PEG₄₈-Fc6 reactions. All fourreaction products displayed a shorter retention time than the adalimumabwhole antibody that further decreased as the size of PEG linkerincreased, confirming the two-handed structure observed by SDS-PAGEanalysis.

The biological activity of the Fab′-cycloalkyne-azide-Fc6 productsevaluated by their ability to neutralize TNF-α-mediated cytotoxicity onmurine WEHI cells treated with actinomycin D. The mouse WEHI-13VAR cellline (ATCC CRL-2148) was obtained from the American Type CultureCollection (Rockville, Md.) and grown in Gibco RPMI media 1640(RPMI-1640) supplemented with 10% fetal bovine serum (FBS) andpenicillin and streptomycin (10 U/ml), obtained from Life Technologies(Grand Island, N.Y.). TNF-α cytotoxity assays were carried out asfollows. WEHI-13VAR cells were plated in 96-well Nunc white cell cultureplates obtained from Thermo Fisher (Waltham, Mass.) at 2×10⁴ cells perwell overnight and then treated with actinomycin D (0.5 μg/ml) obtainedfrom Sigma (St Louis, Mo.) and TNF-α (0.2 ng/ml) in the absence orpresence of TNFR-IgG or other inhibitors. After 24 hr of incubation at37° C./5% CO2, the cell viability was determined with CellTiter-GloLuminescent Cell Viability Assay Systems (Promega, Madison, Wis.)measuring the quantity of the ATP present in metabolically active cellsand luminescence measured using a POLARstar luminometer (BMG LABTECHInc., Cary, N.C.). Each inhibitor was diluted by ten 3-fold serialdilutions starting at 10 μg/ml and measured in duplicate or triplicate.Cytotoxicity data were calculated using the following equations:(1-sample luciferase reading/luciferase reading from cells treated withactinomycin D alone)×100%, and presented as the mean±standard deviation.Enbrel was used as a cytotoxicity positive control and Fc6 as a negativecontrol.

FIG. 23 shows the neutralization of TNF-α-mediated cytotoxicity byFab′-PEG₁₂-Fc6, Fab′-PEG₂₄-Fc6, Fab′-PEG₃₆-Fc6, and Fab′-PEG₄₈-Fc6reaction mixtures compared with the cycloalkyne-modified Fab′ (basedupon an equal amounts of input cycloalkyne-modified Fab′). TheFab′-PEG₁₂-Fc6 and Fab′-PEG₂₄-Fc6 reaction mixtures both displayedcomparable TNF-α neutralization activity compared with that of the inputcycloalkyne-modified Fab′ (upper panel), whereas the Fab′-PEG₃₆-Fc6 andFab′-PEG₄₈-Fc6 reaction mixtures displayed a 1.5-fold and 2.0-foldincrease, respectively, in their TNF-α neutralization activity comparedwith the input cycloalkyne-modified Fab′ (lower panel). Since the amountof two-handed product represented only 10-20% of the totalcycloalkyne-modified Fab′ in each reaction as estimated by SDS-PAGE(FIG. 22), the two-handed products of the Fab′-PEG₃₆-Fc6 andFab′-PEG₄₈-Fc6 reactions are estimated to be at least 7.5-fold and10-fold greater than the input cycloalkyne-modified Fab′, respectively.

Example 3: Fab-Alkyne-Azide-Fc6

Fab-alkyne-azide-Fc6 is prepared by reacting azide-modified Fc6 with analkyne-modified or cycloalkyne-modified Fab protein that is produced bycleavage of an Fab-intein fusion protein as follows. Similarly,Fab-azide-alkyne-Fc6 is prepared by reacting alkyne-modified orcycloalkyne-modified Fc6 with an azide-modified Fab protein that isproduced by cleavage of an Fab-intein fusion protein.

Adalimumab Fab-intein fusion protein is produced by cotransfectingexpression vector pFUSE2ss-DE27-VK-CLIg-hk (SEQ ID NO: 115) withpPUSEss-DE27-Vγ1-CHIg-hG1-Mth-1 (SEQ ID NO: 116) orpFUSEss-DE27-Vγ1-CHIg-hG1-Mth-2 (SEQ ID NO: 117).

Vector pFUSE2ss-DE27-VK-CLIg-hk directs the expression of the pre-kappalight chain of adalimumab shown in SEQ ID NO: 118. Cleavage of theheterologous IL-2 signal sequence by the cellular signal peptidaseprovides the mature kappa light chain of adalimumab shown in SEQ ID NO:119.

Vector pFUSEss-DE27-Vγ1-CHIg-hG1-Mth-1 directs the expression of a firsttype of pre-heavy chain-intein chimeric polypeptide shown in SEQ ID NO:120, in which the adalimumab heavy chain VH and CH1 domains are joinedat their C-terminus to the N-terminus of an RIR1 self-splicing intein atthe autocleavage site. Cleavage of the heterologous IL-2 signal sequenceby the cellular signal peptidase provides the mature heavy chain-inteinfusion protein shown in SEQ ID NO: 121. Together, the proteins of SEQ IDNO: 119 and SEQ ID NO: 121 comprise the adalimumab Fab-1-intein fusionprotein that is secreted into the cell culture fluid.

Vector pFUSEss-DE27-Vγ1-CHIg-hG1-Mth-2 directs the expression of asecond type of pre-heavy chain-intein chimeric polypeptide shown in SEQID NO: 122, in which the adalimumab heavy chain VH and CH1 domains arejoined at their C-terminus to the N-terminus of an RIR1 self-splicingintein at the autocleavage site. Cleavage of the heterologous IL-2signal sequence by the cellular signal peptidase provides the matureheavy chain-intein fusion protein shown in SEQ ID NO: 123. Together, theproteins of SEQ ID NO: 119 and SEQ ID NO: 123 comprise the adalimumabFab-2-intein fusion protein that is secreted into the cell culturefluid.

Protein production is executed by transient expression in CHO-DG44 cellsessentially as described in Example 1, by the cotransfection of SEQ IDNO: 115 with SEQ ID NO: 116 to produce the adalimumab Fab-1-inteinfusion protein, and by cotransfection of SEQ ID NO: 115 with SEQ ID NO:117 to produce adalimumab Fab-2-intein fusion protein.

Alkyne-modified adalimumab Fab proteins are produced by cleavage ofadalimumab Fab-intein fusion proteins with 50 mM cystyl-propargylamideessentially as described in Example 1. The adalimumab Fab-1-inteinfusion protein is cleaved with cystyl-propargylamide to producealkyne-modified adalimumab Fab-1 protein which is a heterodimer proteinof SEQ ID NO: 119 and SEQ ID NO: 124. The adalimumab Fab-2-intein fusionprotein is cleaved with cystyl-propargylamide to produce alkyne-modifiedadalimumab Fab-2 protein which is a heterodimer protein of SEQ ID NO:119 and SEQ ID NO: 125.

Azide-modified adalimumab Fab proteins are produced by cleavage ofadalimumab Fab-intein fusion proteins with 50 mMcystyl-3-azidopropylamide essentially as described in Example 1. Theadalimumab Fab-1-intein fusion protein is cleaved withcystyl-3-azidopropylamide to produce azide-modified adalimumab Fab-1protein which is a heterodimer protein of SEQ ID NO: 119 and SEQ ID NO:126. The adalimumab Fab-2-intein fusion protein is cleaved withcystyl-3-azidopropylamide to produce azide-modified adalimumab Fab-2protein which is a heterodimer protein of SEQ ID NO: 119 and SEQ ID NO:127.

Adalimumab Fab-1-alkyne-azide-Fc6 and Adalimumab Fab-2-alkyne-azide-Fc6are prepared via the reaction of alkyne-modified adalimumab Fab-1protein or alkyne-modified adalimumab Fab-2 protein with Az-DKTHT-Fc6protein (FIG. 6) or Az-PEGx-DKTHT-Fc6 proteins (FIG. 7).

Tris(3-hydroxypropyltriazolylmethyl)amine (THTPA) is prepared asdescribed by Hong et al., Angew. Chem. Int. Ed. 48, 1-7 (2009).Reactions are carried out in 0.1 M sodium phosphate, pH 7.0, with theLinker-Fc at a concentration of 5 mgs/mL or greater, and a molar ratioof >2:1 of Fab-A:Linker-Fc. To the reaction is added a finalconcentration of 0.0001 M CuSO₄, 0.0005 M THTPA. The reaction isinitiated by adding to a final concentration 0.005 M aminoguanidine and0.005 M sodium ascorbate. Following incubation at room temperature for12-18 hours in a closed tube, the reaction mixture is applied to achromatographic column packed with Protein A (GE Lifesciences, NJ) toremove excess reagent and unreacted Fab-A, washed with PBS, eluted with0.1 M Glycine-HCl, pH 2.7, and immediately neutralized by adding 1.0 MTris-HCl, pH 9.0. The eluted Adalimumab Fab-1-alkyne-azide-Fc6 andAdalimumab Fab-2-alkyne-azide-Fc6 products are dialysed against PBS.

Adalimumab Fab-1-azide-alkyne-Fc6 and Adalimumab Fab-2-azide-alkyne-Fc6are prepared via the reaction of azide-modified adalimumab Fab-1 proteinor azide-modified adalimumab Fab-2 protein with cycloalkyne-modified Fc6protein.

Cycloalkyne-modified Fc6 proteins are prepared essentially as describedin Example 1 using DIBAC-PEG₁₂-thioester (Table 1) and otherDIBAC-PEG_(x)-thioesters and DIBAC-PEG_(x)-DKTHT-thioesters similarlyprepared.

DISCUSSION

Aspects of the present invention provides the chemical semisynthesis ofantibodies with nonprotein hinges that incorporate large binding domainssuch as the Fab itself or receptor extracellular domains. The presentinvention relates to the identification of ligation reactions that arecompatible with the native structure and function of the cognateproteins and proceed efficiently. Aspects of the present inventionprovide compounds having nonprotein chains that are both flexible andextendible. Antibody-like molecules provided in embodiments of theinvention have enormous potential as therapeutic candidates withimproved binding affinity for their disease targets.

1-231. (canceled)
 232. A compound having the structure:

wherein C is a polypeptide component of the compound, which polypeptidecomponent has at its N-terminus a sequence of amino acids selected fromthe group consisting of a cysteine, selenocysteine, CP, CPXCP (whereX=P, R, or S) (SEQ ID NOs: 128-130), CDKTHTCPPCP (SEQ ID NO: 131),CVECPPCP (SEQ ID NO: 132), CCVECPPCP (SEQ ID NO: 133) and CDTPPPCPRCP(SEQ ID NO: 134), and comprises consecutive amino acids which (i) areidentical to a stretch of consecutive amino acids present in a chain ofan Fe domain of an antibody; and (ii) bind to an Fc receptor, wherein B″comprises chemical structure B and a terminal reactive group which is anazide or an alkyne; wherein the dashed line between B″ and C representsa peptidyl linkage between B″ and the N-terminus of C.
 233. The compoundaccording to claim 1, wherein the terminal reactive group is a terminalalkyne.
 234. The compound according to claim 1, wherein the terminalreactive group is a cycloalkyne.
 235. The compound according to claim 1,wherein the terminal reactive group is an eight-membered ring.
 236. Thecompound according to claim 1, wherein the terminal reactive group is anazacyclooctyne.
 237. The compound according to claim 1, wherein theterminal reactive group is a biarylazacyclooctyne.
 238. The compoundaccording to claim 1, wherein the terminal reactive group is acyclooctyne.
 239. The compound according to claim 1, wherein thechemical structure B is (a) an organic acid residue or (b) a stretch ofconsecutive amino acid residues which is, or is present in any of thefollowing sequences: EPKSCDKTHTCPPCP, ERKCCVECPPCP,ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3, or ESKYGPPCPSC.
 240. The compoundaccording to claim 2, wherein the chemical structure B is (a) an organicacid residue or (b) a stretch of consecutive amino acid residues whichis, or is present in any of the following sequences: EPKSCDKTHTCPPCP,ERKCCVECPPCP, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3, or ESKYGPPCPSC. 241.The compound according to claim 3, wherein the chemical structure B is(a) an organic acid residue or (b) a stretch of consecutive amino acidresidues which is, or is present in any of the following sequences:EPKSCDKTHTCPPCP, ERKCCVECPPCP, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3, orESKYGPPCPSC.